Peptides for the treatment of cancer

ABSTRACT

The present invention relates to an isolated peptide for use in the treatment of cancer consisting of 12 to 50 amino acid residues comprising: at least two beta-strands, or at least two alpha-helices, or at least one beta-strand and at least one alpha-helix; wherein said beta-strands and/or alpha-helices are preferably separated from each other by at least one turn, wherein the peptide has a net positive charge of +7 or more; wherein said peptide comprises at least one peptide moiety having amino acid sequence (X 1 ) M -X 2 -(X 3 ) P -X 4 -(X 5 ) Q -X 6 -(X 7 ) S  or the reverse sequence thereof, wherein X 1  is a hydrophobic amino acid, preferably selected from the group consisting of phenylalanine (Phe), alanine (Ala), leucine (Leu) and valine (Val), X 2  is a hydrophobic amino acid, preferably tryptophan (Trp), X 3  is selected from the group consisting of alanine (Ala), arginine (Arg), glutamine (Gln), asparagine (Asn), proline (Pro), isoleucine (Ile), leucine (Leu) and valine (Val), X 4  is selected from the group consisting of isoleucine (Ile), phenylalanine (Phe), tryptophan (Trp) and tyrosine (Tyr), X 5  is selected from the group consisting of arginine (Arg), lysine (Lys), tyrosine (Tyr) and phenylalanine (Phe), X 6  is a hydrophobic amino acid, preferably selected from the group consisting of isoleucine (Ile), tryptophan (Trp), valine (Val) and leucine (Leu), and X 7  is selected from the group consisting of arginine (Arg), lysine (Lys), isoleucine (Ile) and serine (Ser), and wherein M is 1 or 2, Q is 1 or 2, P is 2 or 3, and S is 1, 2, 3 or 4 under the proviso that if (X 5 ) Q  is Arg-Arg S is 1; and wherein said peptide is devoid of intramolecular disulfide bonds.

The present application is a continuation from prior U.S. applicationSer. No. 14/760,445 filed Jul. 10, 2015, which is a 371 application ofPCT/EP2014/050330 filed Jan. 9, 2014, which claims priority to EPApplication No. 13150974.7 filed Jan. 11, 2013. The entire contents ofeach of the above-referenced disclosures are specifically incorporatedby reference herein without disclaimer.

The present invention relates to an isolated peptide exhibitingantitumoral effects.

Every year millions of people are diagnosed with cancer worldwide.Notwithstanding in the last decades much progress has been achieved incancer therapy, nevertheless cancer remains a leading cause of death.Nowadays, surgery, chemotherapy, radiation, hormone ablation therapy andtargeted therapy are the standard treatments, but in the year 2008 thesewere not curative in more than 50% of the cases. Furthermore, the use ofthese types of therapy is limited due to resistance and is accompaniedby potential toxicity and diverse side effects due to inadequatespecificity for tumor cells. Obviously, the discovery of new and morespecific targets, together with the design of specific antitumor drugs,is one of the major interests in cancer research.

Cancer cells are often well characterized, but little is known about theplasma cell membrane, or to be more precise, the arising differences inthe lipid composition in carcinogenesis. Eukaryotic plasma membranesusually comprise an overall neutral charge on the outer leaflet due tothe zwitterionic phosphatidylcholine (PC) and sphingomyelin (SM). Thenegatively charged phospholipid phosphatidylserine (PS) together withthe major part of phosphatidylethanolamine (PE) normally only assemblesin the inner leaflet of eukaryotic plasma membranes. This asymmetricdistribution of phospholipids is well documented and is maintained by anATP-dependent aminophospholipid translocase. This asymmetry can get lostdue to exposure of the negatively charged phosphatidylserine on thesurface of cancerous and other pathological cells, apoptotic cells, aswell as platelets and erythrocytes upon activation.

Based on the knowledge of PS exposure, new strategies for the design ofanticancer drugs can be considered, especially cationic host defensederived peptides interacting with negatively charged phospholipids. Hostdefense peptides have emerged as potential alternative anticancertherapeutics offering many advantages over other therapies (Mader etal., Exp. Op. Investig. Drugs 15 (2006), 933-946). Because of their modeof action and specificity—the cell membrane being the majortarget—resistance and cytotoxicity are less likely to occur and thus,they are also expected to cause fewer side effects. Furthermore, thesepeptides mostly damage cell membranes within minutes, which would hinderformation of resistance. Host defense peptides being part of the innateimmune system of many diverse species (e.g. mammals, insects,amphibians) were initially discovered because of their antimicrobialactivity. Currently, the antimicrobial peptide database lists more than100 natural host defense peptides with antitumor activity. Examples forantimicrobial peptides are disclosed in WO 2008/002165 A1.

One prominent member of anticancer peptides is bovine lactoferricin(bLFcin), which is generated from lactoferrin through pepsin cleavage.bLFcin possesses an acyclic twisted antiparallel β-sheet structure dueto a disulfide bridge between two cysteine residues. This peptide isable to inhibit liver and lung metastasis in mice. In vivo studies withbLFcin on fibrosarcoma, melanoma and colon carcinoma tumors revealedmassive necrosis of the tumor tissue after exposure to the peptide (Yooet al. Jpn. J Cancer Res. 88 (1997):184-190). Furthermore, it is knownthat bLFcin inhibits the tumor growth of neuroblastoma xenografts innude rats. Clarification of the mechanism revealed that bLFcin inducesapoptosis in human tumor cells through a pathway mediated by productionof the intracellular ROS and activation of Ca²⁺/Mg²⁺-dependentendonucleases.

It is an object of the present invention to provide compounds andpreparations which can be used to treat cancer.

Therefore the present invention relates to an isolated peptide to beused in the treatment of cancer consisting of 12 to 50 amino acidresidues comprising

-   -   at least two beta-strands, or    -   at least two alpha-helices or    -   at least one beta-strand and at least one alpha-helix,    -   wherein said beta-strands and/or alpha-helices are preferably        separated from each other by at least one turn, wherein the        peptide has a net positive charge of +7 or more;    -   wherein said peptide comprises at least one peptide moiety        having amino acid sequence        (X₁)_(M)-X₂-(X₃)_(P)-X₄-(X₅)_(Q)-X₆-(X₇)_(S) or the reverse        sequence thereof, wherein        -   X₁ is a hydrophobic amino acid, preferably selected from the            group consisting of phenylalanine (Phe), alanine (Ala),            leucine (Leu) and valine (Val),        -   X₂ is a hydrophobic amino acid, preferably tryptophan (Trp),        -   X₃ is selected from the group consisting of alanine (Ala),            arginine (Arg), glutamine (Gln), asparagine (Asn), proline            (Pro), isoleucine (Ile), leucine (Leu) and valine (Val),        -   X₄ is selected from the group consisting of isoleucine            (Ile), phenylalanine (Phe), tryptophan (Trp) and tyrosine            (Tyr),        -   X₅ is selected from the group consisting of arginine (Arg),            lysine (Lys), tyrosine (Tyr) and phenylalanine (Phe),        -   X₆ is a hydrophobic amino acid, preferably selected from the            group consisting of isoleucine (Ile), tryptophan (Trp),            valine (Val) and leucine (Leu), and        -   X₇ is selected from the group consisting of arginine (Arg),            lysine (Lys), isoleucine (Ile) and serine (Ser), and wherein        -   M is 1 or 2,        -   Q is 1 or 2,        -   P is 2 or 3, and        -   S is 1, 2, 3 or 4 under the proviso that if (X₅)_(Q) is            Arg-Arg S is 1; and    -   wherein said peptide is devoid of intramolecular disulfide        bonds,        preferably, said peptide being selected from the group        consisting of PFWRIRIRRXRRIRIRWFP (SEQ ID. No. 128),        PWRIRIRRXRRIRIRWP (SEQ ID No. 184), RWKRINRQWFFWQRNIRKWR (SEQ        ID. No. 106), PFWRIRIRRPFWRIRIRR (SEQ ID. No. 125),        PFFWRIRIRRPFFWRIRIRR (SEQ ID. No. 141) and PFWRIRIRRRRIRIRWFP        (SEQ ID. No. 127), especially SEQ ID. Nos. 128, 184 and 106,        wherein “X” is proline (Pro) and/or glycine (Gly)₁₋₃.

It turned out that peptides having a net positive charge of +7 andcomprising at least two beta-strands or at least two alpha-helices or atleast one beta-strand and at least one alpha-helix and are preferablyseparated by at least one turn exhibit cytotoxic effects oncancerous/tumor cells in mammals. This means that the peptides of thepresent invention are able to affect the viability of such cells leadingto their destruction. The cytotoxic effects of the peptides of thepresent invention are highly specific for cancerous/tumor cells. Thismeans that these peptides affect healthy cells to a much lower extent(preferably to at least 10%, more preferably to at least 20%, even morepreferably to at least 50%, in particular to at least 90 to 100%)compared to cancerous/tumor cells. This high specificity of the peptidesof the present invention allows treating mammals, in particular humans,with a much higher efficacy reducing commonly known side-effectsregularly described for anti-cancer compounds. The cytotoxic effect ofsuch compounds is usually unspecific resulting in the destruction notonly of cancerous/tumor cells but also of healthy cells.

The antitumour properties of the peptides according to the presentinvention are specifically surprising, because the present peptides are,by their definition, devoid of disulfide bonds, i.e. devoid ofintramolecular S—S bonds (due to the absence of (at least) two cysteineresidues in the amino acid sequence that form this bond). The moleculesof the present invention have been derived from human lactoferricin(hLFcin: TKCFQWQRNMRKVRGPPVSCIKRDS (SEQ ID No. 207)), a peptide thatcontains two cysteins that form an intramolecular disulfide bond. One ofthe staring compounds of the developments of the present invention was apart of hLFcin that lacks the cysteins and wherein the methionine hasbeen replaced by isoleucine (FQWQRNIRKVR; SEQ ID NO. 87; “PEP parent”).In contrast to other membrane active peptides, PEP parent therefore wasdevoid of intramolecular disulfide bonds (and even devoid of cysteineresidues) that were thought to be important for membrane active(antimicrobial) function (see e.g. Harwig et al., Eur. J. Biochem. 240(1996), 352-357). In fact, eliminating the disulfide bonds from suchmolecules significantly reduced membrane permeabilising activity of suchpeptides (Matsuzaki et al., Biochemistry 32 (1993), 11704-11710;Tamamura et al., Chem. Pharm. Bull. 43 (5) (1995), 853-858). It wasknown to a person with average skill in the art that disulfide bonds areof significant importance for membrane active peptides. It was thereforesurprising that the peptides according to the present invention that arealso membrane active, do not contain intramolecular disulfide bonds. Infact, the preferred peptide moieties which are responsible for theantitumor effect of the peptides according to the present invention arecompletely free of cysteine residues. The antitumour peptide accordingto the present invention is therefore usually cysteine-free. Cysteineresidues can, however, if necessary, be used to couple the presentpeptide to other molecules (e.g. to carriers (e.g. carrier proteins))which are preferably released from the peptides according to the presentinvention before administration or (after administration) in the body ofa patient. In such cases, the disulfide bond is not located within thepeptide defined by the present invention but between the peptideaccording to the present invention (that is defined by a continuouspeptide bond connection (amino acid sequence)) and another chemicalcompound. That the peptides according to the present invention aredevoid of intramolecular disulfide bonds is specifically surprising withregard to the fact that the peptides according to the present inventionare derived from human lactoferricin (hLFcin) for which the disulfidebond has been disclosed as being essential for the antimicrobialactivity and also held relevant for the antitumor activity (Gifford etal., Cell. Mol. Life Sci. 62 (2005), 2588-2598). Also for bovinelactoferricin (LFcinB), the disulfide bond was regarded as necessary forthe antitumoral activity (Eliassen et al., Antican. Res. 22 (2002),2703-2710).

This also shows that the antitumor activity of the peptides of thepresent invention is not dependent on a specifically stabilised (i.e. bydisulfide bonds) secondary structure, but that the beta-strand andalpha-helix folding is sufficient for the peptides defined e.g. by theconsensus sequences of the peptide moieties present in the peptideaccording to the present invention.

Preferably, the isolated peptides according to the present inventioncontain at least one turn. A “turn” is an element of secondary structurein polypeptides where the polypeptide chain reverses its overalldirection. A “turn” may, in a structurally more precise manner, bedefined as a “structural motif where the Cα atoms of two residuesseparated by few (usually 1 to 5) peptide bonds are in close approach(<7 Å), while the corresponding residues do not form a regular secondarystructure element such as an alpha helix or beta sheet”. The turnaccording to the present invention may consist also of amino acids fromthe peptide moieties (specifically, of course, if no separate linker islocated between the moieties). However, the turn according to thepresent invention may also be a loop (an “ω-loop being a catch-all termfor a longer, extended or disordered loop without fixed internalhydrogen bonding; see also Toniolo et al., CRC Crit. Rev. Biochem. 9(1980): 1-44).

According to the present invention the at least two beta-strands or atleast two alpha-helices or the at least one beta-strand or the at leastone alpha-helix are preferably separated by at least one turn resultingin peptides with alpha-helix and/or beta-strand moieties having thefollowing general basic structures:

-   -   a) beta-strand-(turn)-beta-strand    -   b) alpha-helix-(turn)-alpha-helix    -   c) beta-strand-(turn)-alpha-helix    -   d) alpha-helix-(turn)-beta-strand

According to the present invention the peptides disclosed herein mayalso comprise 3, 4 or even 5 beta-strands or alpha-helices as peptidemoieties. In such a case the beta-strands or alpha-helices of thepeptide can be grouped (e.g. two beta strands are located adjacent toeach other) and preferably separated by one or more (e.g. 2, 3 or 4)turns or every single strand or helix is preferably separated by one ormore turns. The isolated peptide of the present invention may thereforecomprise also more than one stretches having the above general basicstructure.

The peptides of the present invention have a net positive charge of +7or more (for the purpose of a formal definition for the presentinvention, the net positive charge can be regarded as being defined atpH 7.4 in PBS buffer (phosphate buffered saline: 20 mM NaPi, 130 mMNaCl, pH 7.4)). This means that the peptides of the present inventionmay have preferably a net positive charge of +8, +9, +10, +11, +12, +13,+14, +15 or even of +20. A net positive charge of at least +7 of thepeptides of the present invention results in a better adsorption to thetarget membrane (negatively charged) and better stabilization of thesecondary structure by hydrogen bridge bonds. Calculation of net chargesof a peptide is performed by adding the positive charges in apolypeptide (and, if present, subtracting the negative net charges). Forexample, a peptide with three lysine and four arginine residues has anet positive charge of +7 at a pH below 10.5 (pK lysine ˜10.5; argininewould even be positively charged at pH 12.5!). Amidating the C-terminalcarboxylate group of a peptide adds one additional positive charge (seee.g. Yang et al., J. Pep. Sci. 10 (2004), 37-46). Another possibility toarrive at the net charge Z of a peptide at a certain pH can be estimatedby calculation

$Z = {{\sum\limits_{i}^{\;}{{Ni}\frac{10^{pKai}}{10^{p\; H} + 10^{pKai}}}} - {\sum\limits_{i}^{\;}{{Ni}\frac{10^{p\; H}}{10^{p\; H} + 10^{pKai}}}}}$where N_(i) are the number, and pKa_(i) the pKa values, of theN-terminus and the side chains of Arginine, Lysine, and Histidine. Thej-index pertain to the C-terminus and the Aspartic Acid, Glutamic Acid,Cysteine, Tyrosine amino acids.

The present peptides are preferably designed as “membrane activepeptides”, i.e. peptides that have—due to their physicochemicalproperties—an affinity to membranes. Common properties of membraneactive peptides are disclosed e.g. in Last et al., Protein Science 22(2013), 870-882 or Wang et al. J. Biol. Chem. 114 (2010), 13726-13735).Membrane active peptides are able to perturb the structural barrierfunction of cell membranes, which may eventually lead to cell lysis andcell death; these peptides share two common features: amphipathicity anda net positive charge (see Rekdal at al., Journal of BiologicalChemistry 287 (2012), 233-244). They share common features as cationicresidues being reported to be important for the initial electrostaticinteraction and hydrophobic residues being important for membranedisruption (see Lohner et al., Combinatorial Chemistry & High ThroughputScreening, 2005, 8, 241-256). Presence of membrane active properties canbe determined by a variety of methods. Preferred methods for verifyingmembrane active property according to the present invention arepermeability studies (dye release) using liposomes composed ofphosphatidylserine, naturally exposed by cancer cells (Riedl et al., BBA1808 (2011) 2638-2645) and thus relevant for cancer cells, as well asmembrane permeabilization of tumor cells (propidium iodide (PI)-uptake).A membrane active peptide, especially a cancer membrane active peptideof the present invention should therefore preferably exhibit at least amembrane permeabilizing activity (ANTS/DPX-release) on PS-liposomes ofmore than 20% at a peptide concentration of 8 μM (test according toZweytick et al., J. Biol. Chem. 286 (2011), 21266-21276). The PI-uptakeof 10⁵ tumor cells should preferably be at least 20% after 8 hours ofpeptide incubation at a concentration of 20 μM of the membrane activepeptide (test according to Schröder-Borm et al., J. FEBS Lett. 2005 Nov.7; 579(27):6128-34).

Alpha helix (α-helix) is a common motif in the secondary structure ofproteins, polypeptides and peptides. Alpha helices have a right-handedcoiled or spiral conformation, in which every backbone N—H group donatesa hydrogen bond to the backbone C═O group of the amino acid fourresidues earlier. The beta sheet (β-sheet) is the second form of regularsecondary structure in proteins, polypeptides and peptides. Beta-sheetsconsist of beta strands connected laterally by at least two or threebackbone hydrogen bonds, forming a generally twisted, pleated sheet. Aturn is a structural motif where the Cα atoms of two residues separatedby one or more peptide bonds are in close approach (approx. <7 Å), whilethe corresponding residues do not form a regular secondary structureelement such as an alpha-helix or beta-sheet (Chou P Y et al., AnnualReview of Biochemistry 47 (1978):251-276). The secondary structure ofputative membrane active isolated peptides can accurately be predictedby the online program PEP-FOLD: E.g.http://bioserv.rpbs.univ-paris-diderot.fr/PEP-FOLD/ (see Maupetit etal., Nucleic Acids Res. 37 (2009), W498-W503 and/or Thévenet et al.,Nucleic Acid Res. 40 (2012), W288-W293).

The person skilled in the art is able to identify peptides exhibitingthe properties as described herein using known methods. The secondarystructure can be identified as described above. The net charge can becalculated by summing up the positive and negative charges of the aminoacid residues present in a peptide.

One skilled in the art can easily synthesize the peptides of the presentinvention. Standard procedures for preparing synthetic peptides are wellknown in the art. Peptides of the present invention can be synthesizedby commonly used methods as t-BOC or FMOC protection, preferably FMOCprotection, of alpha-amino groups. Both methods involve stepwisesyntheses whereby a single amino acid is added at each step startingfrom the carboxyl-terminus of the peptide (See, Coligan et al., CurrentProtocols in Immunology, Wiley Interscience, 2002, Unit 9). Peptides ofthe invention can also be synthesized by the solid phase peptidesynthesis methods well known in the art. (Merrifield, J. Am. Chem. Soc.,85:2149, 1963), and Stewart and Young, Solid Phase Peptides Synthesis,(1969). Peptides can be synthesized using acopoly(styrene-divinylbenzene) containing 0.1-1.0 mMol amines/g polymer.On completion of chemical synthesis, the peptides can be deprotected andcleaved from the polymer by treatment with liquid HF-10% anisole forabout 0.25 to 1 hour at 0° C. After evaporation of the reagents, thepeptides are extracted from the polymer with 1% acetic acid solutionwhich is then lyophilized to yield the crude material. This cantypically be purified by such techniques as gel filtration on SephadexG-15 using 5% acetic acid as a solvent, by high pressure liquidchromatography, and the like. Lyophilization of appropriate fractions ofthe column will yield the homogeneous peptide or peptide derivatives,which can then be characterized by such standard techniques as aminoacid analysis, thin layer chromatography, high performance liquidchromatography, ultraviolet absorption spectroscopy, molar rotation,solubility, and assessed by the solid phase Edman degradation (see e.g.Protein Purification, M. P. Deutscher, ed. Methods in Enzymology, Vol182, Academic Press, 1990). Automated synthesis using FMOC solid phasesynthetic methods can be achieved using an automated peptide synthesizer(Model 432A, Applied Biosystems, Inc.).

The peptides/polypeptides of the present invention can also besynthesized using a fusion protein microbial method in which an anioniccarrier peptide is fused to a cationic peptide. A method for suchmicrobial production of cationic peptides having anti-microbial activityis provided in U.S. Pat. No. 5,593,866.

The peptides of the present invention thus produced can be purified byisolation/purification methods for proteins generally known in the fieldof protein chemistry. More particularly, there can be mentioned, forexample, extraction, recrystallization, salting out with ammoniumsulfate, sodium sulfate, etc., centrifugation, dialysis,ultrafiltration, adsorption chromatography, ion exchange chromatography,hydrophobic chromatography, normal phase chromatography, reversed-phasechromatography, gel filtration method, gel permeation chromatography,affinity chromatography, electrophoresis, countercurrent distribution,etc. and combinations of these. Most effective is a method byreversed-phase high performance liquid chromatography.

The peptides of the present invention may form a salt by addition of anacid. Examples of the acid include inorganic acids (such astrifluoroacetic acid, hydrochloric acid, hydrobromic acid, phosphoricacid, nitric acid, and sulfuric acid) or organic carboxylic acids (suchas acetic acid, propionic acid, maleic acid, succinic acid, malic acid,citric acid, tartaric acid, and salicylic acid), acidic sugars such asglucuronic acid, galacturonic acid, gluconic acid, ascorbic acid, etc.,acidic polysaccharides such as hyaluronic acid, chondroitin sulfates,alginic acid, or organic sulfonic acids (such as methanesulfonic acid,and p-toluenesulfonic acid), and the like. Of these salts, preferred isa pharmaceutically acceptable salt.

The peptides of the present invention may form a salt with a basicsubstance. Examples of the salt include, for example, pharmaceuticallyacceptable salts selected from salts with inorganic bases such as alkalimetal salts (sodium salt, lithium salt, potassium salt etc.), alkalineearth metal salts, ammonium salts, and the like or salts with organicbases, such as diethanolamine salts, cyclohexylamine salts and the like.

The term “amino acid” and “amino acid residue” as used herein meansL-amino acids. However, also D-amino acids may be employed in themanufacturing of the peptides according to the present invention.

The peptide of the present invention preferably exhibits amphipathicproperties. This means that the peptide of the present invention maycomprise hydrophobic and hydrophilic regions. Methods to determineamphipathic properties are well known in the art.

According to the present invention the isolated peptide comprises atleast one peptide moiety having amino acid sequence(X₁)_(M)-X₂-(X₃)_(P)-X₄-(X₅)_(Q)-X₆-(X₇)_(S) (SEQ ID No. 203) or thereverse sequence thereof, wherein

-   -   X₁ is a hydrophobic amino acid, preferably selected from the        group consisting of phenylalanine (Phe), alanine (Ala), leucine        (Leu) and valine (Val),    -   X₂ is a hydrophobic amino acid, preferably tryptophan (Trp),    -   X₃ is selected from the group consisting of alanine (Ala),        arginine (Arg), glutamine (Gln), asparagine (Asn), proline        (Pro), isoleucine (Ile), leucine (Leu) and valine (Val),    -   X₄ is selected from the group consisting of isoleucine (Ile),        phenylalanine (Phe), tryptophan (Trp) and tyrosine (Tyr),    -   X₅ is selected from the group consisting of arginine (Arg),        lysine (Lys), tyrosine (Tyr) and phenylalanine (Phe),    -   X₆ is a hydrophobic amino acid, preferably selected from the        group consisting of isoleucine (Ile), tryptophan (Trp), valine        (Val) and leucine (Leu), and    -   X₇ is selected from the group consisting of arginine (Arg),        lysine (Lys), isoleucine (Ile) and serine (Ser), and wherein    -   M is 1 or 2,    -   Q is 1 or 2,    -   P is 2 or 3, and    -   S is 1, 2, 3 or 4 under the proviso that if (X₅)_(Q) is Arg-Arg        S is 1.

As used herein the term “reverse sequence” of an amino acid sequencemeans that a specific sequence is reversed. For instance, the reversesequence of the amino acid sequence ABCDEFG is GFEDCBA.

The at least one peptide moiety has preferably an amino acid sequenceselected from the group consisting of FWQRIRKVR (SEQ ID No. 1),FWQRRIRKVRR (SEQ ID No. 2), FWQRKIRKVRK (SEQ ID No. 3), FWQRNIRIRR (SEQID No. 4), FWQRNIRKVR (SEQ ID No. 5), FWQRNIRVR (SEQ ID No. 6),FWQRNIRKVRR (SEQ ID No. 7), FWQRNIRKVKK (SEQ ID No. 8), FWQRNIRKVRRR(SEQ ID No. 9), FWQRNIRKVKKK (SEQ ID No. 10), FWQRNIRKVRRRR (SEQ ID No.11), FWQRNIRKVRRRI (SEQ ID No. 12), FWQRNIRKVKKKK (SEQ ID No. 13),FWQRNIRKVKKKI (SEQ ID No. 14), FWQRNIRKIR (SEQ ID No. 15), FWQRNIRKLR(SEQ ID No. 16), FWQRNIRKWR (SEQ ID No. 17), FWQRNWRKVR (SEQ ID No. 18),FWQRNFRKVR (SEQ ID No. 19), FWQRNYRKVR (SEQ ID No. 20), FWQRNIRKVS (SEQID No. 21), FWQRRIRIRR (SEQ ID No. 22), FWQRPIRKVR (SEQ ID No. 23),FWQRRIRKWR (SEQ ID No. 24), FWPRNIRKVR (SEQ ID No. 26), FWARNIRKVR (SEQID No. 27), FWIRNIRKVR (SEQ ID No. 28), FWLRNIRKVR (SEQ ID No. 29),FWVRNIRKVR (SEQ ID No. 30), FWQRNIFKVR (SEQ ID No. 31), FWQRNIYKVR (SEQID No. 32), FAWQRNIRKVR (SEQ ID No. 33), FLWQRNIRKVR (SEQ ID No. 35) andFVWQRNIRKVR (SEQ ID No. 36) or the reverse sequence thereof.

According to another preferred embodiment of the present invention theisolated peptide comprises at least one peptide moiety having amino acidsequence(X_(1′))_(M′)-X_(2′)-(X_(3′))_(P′)-(X_(4′))_(Q′)-(X_(5′))_(T′)-(X_(6′))_(R′)-(X_(7′))_(S′)(SEQ ID No. 204) or the reverse sequence thereof, wherein

-   -   X_(1′) is a hydrophobic amino acid, preferably selected from the        group consisting of phenylalanine (Phe) and isoleucine (Ile),    -   X_(2′) is a hydrophobic amino acid, preferably tryptophan (Trp),    -   X_(3′) is selected from the group consisting of glycine (Gly),        asparagine (Asn), isoleucine (Ile) and phenylalanin (Phe),    -   X_(4′) is isoleucine (Ile) or tryptophan (Trp),    -   X_(5′) is arginine (Arg) or lysine (Lys),    -   X_(6′) is a hydrophobic amino acid, preferably selected from the        group consisting of isoleucine (Ile), tryptophan (Trp) and        valine (Val) and    -   X_(7′) is arginine (Arg), and wherein    -   M′ is 1 or 2,    -   T′ is 1 or 2,    -   R′ is 0 or 1,    -   P′ is 1, 2 or 3,    -   Q′ is 1, and    -   S′ is 0, 1 or 2.

The at least one peptide moiety in the peptide of the present inventionmay have an amino acid sequence selected from the group consisting ofFWRIRKWR (SEQ ID No. 37), FWRIRKVR (SEQ ID No. 38), FWRWRR (SEQ ID No.39), FWRRWRR (SEQ ID No. 40), FWRRWIRR (SEQ ID No. 41), FWRGWRIRR (SEQID No. 42), FWRRFWRR (SEQ ID No. 43), FWRWRWR (SEQ ID No. 44), FWRIWRWR(SEQ ID No. 45), FWRIWRIWR (SEQ ID No. 46), FWRNIRKWR (SEQ ID No. 47)and FWRRRIRIRR (SEQ ID No. 48) or the reverse sequence thereof.

According to a further preferred embodiment of the present invention theisolated peptide comprises at least one peptide moiety having amino acidsequence(X_(1″))_(M″)-X_(2″)-(X_(3″))_(P″)-(X_(4″))_(Q″)-(X_(5″))_(R″)-(X_(6″))_(S″)(SEQ ID No. 205) or the reverse sequence thereof, wherein

-   -   X_(1″) is a hydrophobic amino acid, preferably selected from the        group consisting of proline (Pro) and phenylalanine (Phe),    -   X_(2″) is a hydrophobic amino acid, preferably tryptophan (Trp),    -   X_(3″) is selected from the group consisting of alanine (Ala),        arginine (Arg), glutamine (Gln), lysine (Lys), tryptophan (Trp)        and isoleucine (Ile),    -   X_(4″) is selected from the group consisting of arginine (Arg)        and aspartate (Asp),    -   X_(5″) is a hydrophobic amino acid, preferably selected from the        group consisting of isoleucine (Ile), tryptophan (Trp),        phenylalanine (Phe), valine (Val) and leucine (Leu), and    -   X_(6″) is selected from the group consisting of arginine (Arg),        lysine (Lys), isoleucine (Ile), serine (Ser) and aspartate        (Asp), and wherein    -   M″ is 0, 1, 2 or 3,    -   Q″ is 0, 1, 2 or 3,    -   R″ is 1 or 2,    -   P″ is 1, 2 or 3, and    -   S″ is 1, 2 or 3.

The at least one peptide moiety has preferably an amino acid sequenceselected from the group consisting of PFWRWRIWR (SEQ ID No. 50),PFWRIRIRR (SEQ ID No. 51), PFWRQRIRR (SEQ ID No. 52), PFWRARIRR (SEQ IDNo. 53), PFWRKRIRR (SEQ ID No. 54), PFWRKRLRR (SEQ ID No. 55), PFWRKRWRR(SEQ ID No. 56), PFWRRRIRR (SEQ ID No. 57), PFWRRRWRR (SEQ ID No. 58),PFWRIRIRRD (SEQ ID No. 59), PFFWRIRIRR (SEQ ID No. 60), PWRIRIRR (SEQ IDNo. 61), PFWRRQIRR (SEQ ID No. 81), PFWRKKLKR (SEQ ID No. 82), PWRRIRR(SEQ ID No. 83), PWRRKIRR (SEQ ID No. 84) and PFWRRIRIRR (SEQ ID No. 85)or the reverse sequence thereof.

According to a preferred embodiment of the present invention theisolated peptide comprises at least one peptide moiety having amino acidsequence(X_(1′″))_(M′″)-(X_(2′″))_(O′″)-X_(3′″)-(X_(4′″))_(P′″)-(X_(5′″))_(Q′″)-(X_(6′″))_(T′″)-(X_(7′″))_(R′″)-(X_(8′″))_(S′″)(SEQ ID No. 206) or the reverse sequence thereof, wherein

-   -   X_(1′″) is a hydrophobic amino acid, preferably selected from        the group consisting of proline (Pro) and phenylalanine (Phe),    -   X_(2′″) is a basic amino acid, preferably arginine (Arg),    -   X_(3′″) is a hydrophobic amino acid, preferably tryptophan        (Trp),    -   X_(4′″) is selected from the group consisting of alanine (Ala),        arginine (Arg), glutamine (Gln), asparagine (Asn) and lysine        (Lys),    -   X_(5′″) is selected from the group consisting of isoleucine        (Ile), phenylalanine (Phe) and tryptophan (Trp),    -   X_(6′″) is selected from the group consisting of glutamine        (Gln), arginine (Arg) and asparagine (Asn),    -   X_(7′″) is a hydrophobic amino acid, preferably selected from        the group consisting of isoleucine (Ile), tryptophan (Trp) and        phenylalanine (Phe), and    -   X_(8′″) is arginine (Arg), and wherein    -   M′″ is 0, 1, 2 or 3,    -   T′″ is 0, 1, 2 or 3,    -   O′″ is 0 or 1,    -   P′″ is 1, 2 or 3,    -   Q′″ is 1 or 2, and    -   R′″ and S′″ are 0, 1 or 2.

The at least one peptide may have an amino acid sequence selected fromthe group consisting of FWRNIRIRR (SEQ ID No. 72), FWQRIRIRR (SEQ ID No.73), FWRWRIWR (SEQ ID No. 74), FWRIRIRR (SEQ ID No. 75), FWRNIRIWRR (SEQID No. 76) and FWRNIRIRR (SEQ ID No. 77) or the reverse sequencethereof.

The isolated peptide comprises preferably at least one peptide moietyhaving an amino acid sequence selected from the group consisting ofRFWQRNIRKVRR (SEQ ID No. 62), RFWQRNIRKYR (SEQ ID No. 63), PFWQRNIRKWR(SEQ ID No. 64), RFRWQRNIRKYRR (SEQ ID No. 65), RWKRINRQWF (SEQ ID No.66), KRFCFKK (SEQ ID No. 67), KRFSFKKC (SEQ ID No. 68), KRWSWKK (SEQ IDNo. 69), FRFSFKK (SEQ ID No. 70), RRFWFRR (SEQ ID No. 71), RFWQRNIRIRR(SEQ ID No. 78), RWQRNIRIRR (SEQ ID No. 79) and RRWFWRR (SEQ ID No. 86)or the reverse sequence thereof.

According to a further embodiment of the present invention the isolatedpeptide comprises at least one peptide moiety having an amino acidsequence selected from the group consisting of FIWQRNIRKVR (SEQ ID No.34), FIWRWRWR (SEQ ID No. 49) and RRIRINRQWF (SEQ ID No. 80) or thereverse sequence thereof.

The isolated peptide of the present invention may comprise a singlepeptide moiety or the reverse sequence thereof as defined above.However, the isolated peptide may also comprise a multiplicity (at leasttwo, at least three, at least four etc.) of said single peptide moietiesor peptide moieties having the reversed sequence thereof. According to avery particular preferred embodiment of the present invention theisolated peptide comprises at least two, most preferably two, of theaforementioned peptide moieties with the sequence(X₁)_(M)-X₂-(X₃)_(P)-X₄-(X₅)_(Q)-X₆-(X₇)_(S) or the reverse sequencethereof.

According to a particularly preferred embodiment of the presentinvention the peptide of the present invention is selected from thepeptides as shown in Table 1 having at least two beta-strands, or atleast two alpha-helices or at least one beta-strand and at least onealpha-helix, said beta-strands and/or alpha-helices optionally beingseparated from each other by at least one turn. Preferred are thosepeptides having an amino acid sequence selected from the groupconsisting of SEQ ID. No. 89, SEQ ID. No. 91, SEQ ID. No. 93, SEQ ID.No. 95, SEQ ID. No. 97, SEQ ID. No. 98, SEQ ID. No. 99, SEQ ID. No. 101,SEQ ID. No. 103, SEQ ID. No. 105, SEQ ID. No. 106, SEQ ID. No. 107, SEQID. No. 109, SEQ ID. No. 111, SEQ ID. No. 114, SEQ ID. No. 116, SEQ ID.No. 117, SEQ ID. No. 118, SEQ ID. No. 120, SEQ ID. No. 122, SEQ ID. No.123, SEQ ID. No. 124, SEQ ID. No. 125, SEQ ID. No. 126, SEQ ID. No. 127,SEQ ID. No. 128, SEQ ID. No. 129, SEQ ID. No. 130, SEQ ID. No. 131, SEQID. No. 132, SEQ ID. No. 134, SEQ ID. No. 136, SEQ ID. No. 137, SEQ ID.No. 138, SEQ ID. No. 139, SEQ ID. No. 140, SEQ ID. No. 141, SEQ ID. No.142, SEQ ID. No. 143, SEQ ID. No. 144, SEQ ID. No. 145, SEQ ID. No. 146,SEQ ID. No. 148, SEQ ID. No. 149, SEQ ID. No. 150, SEQ ID. No. 156, SEQID. No. 153, SEQ ID. No. 154, SEQ ID. No. 156, SEQ ID. No. 158, SEQ ID.No. 160, SEQ ID. No. 162, SEQ ID. No. 164, SEQ ID. No. 166, SEQ ID. No.168, SEQ ID. No. 170, SEQ ID. No. 172, SEQ ID. No. 174, SEQ ID. No. 176,SEQ ID. No. 178, SEQ ID. No. 180, SEQ ID. No. 181, SEQ ID. No. 182, SEQID. No. 183, SEQ ID. No. 184, SEQ ID. No. 186, SEQ ID. No. 188, SEQ ID.No. 190, SEQ ID. No. 192, SEQ ID. No. 194, SEQ ID. No. 195, SEQ ID. No.197, SEQ ID. No. 199, SEQ ID. No. 200, SEQ ID. No. 201 and SEQ ID. No.202. The variable “X” within these sequences can be 1 to 3 (i.e. 1, 2 or3) glycine or proline residues, preferably 1 proline residue.Particularly preferred peptides are those having a sequence selectedfrom the group consisting of SEQ ID. No. 125, SEQ ID. No. 126, SEQ ID.No. 127, SEQ ID. No. 128, SEQ ID. No. 105, SEQ ID. No. 106, SEQ ID. No.107, SEQ ID. No. 174, SEQ ID. No. 176, SEQ ID. No. 141, SEQ ID. No. 142,SEQ ID. No. 143, SEQ ID. No. 144, SEQ ID. No. 181, SEQ ID. No. 182, SEQID. No. 183 and SEQ ID. No. 184.

According to a preferred embodiment of the present invention the peptideof the present invention has/comprises/consists of a sequence selectedfrom the group consisting of SEQ ID. No. 125, SEQ ID. No. 127, SEQ ID.No. 128, SEQ ID. No. 106 and SEQ ID. No. 141 and No. 184. For example,SEQ ID. Nos. 128, 184 and 106, especially Nos. 128 and 184, arespecifically suitable for cancer treatment, especially for the treatmentof glioblastoma and malignant melanoma.

Of course the isolated peptide of the present invention may alsocomprise a combination of at least two of the aforementioned peptides.

According to a preferred embodiment of the present invention at leasttwo peptides having an amino acid sequence as defined herein or thereverse sequence thereof are fused directly or via a linker, whereinsaid linker is preferably part of the turn, to each other.

According to a further preferred embodiment of the present inventionsaid isolated peptide comprises at least two, preferably two, peptidemoieties with the sequence (X₁)_(M)-X₂-(X₃)_(P)-X₄-(X₅)_(Q)-X₆-(X₇)_(S)or the reverse sequence thereof (“retro” sequence) having the same aminoacid sequence and being selected from the peptide moieties having anamino acid sequence as defined above or the reverse sequence thereof,wherein the at least two peptide moieties are fused directly or via alinker to each other (e.g. the “reverse” or “retro” sequence of X₁X₂X₃X₄is X₄X₃X₂X₁; e.g. “retro” sequence of PWRIRIRR is RRIRIRWP).

Said isolated peptide may comprise at least two, preferably two, peptidemoieties, wherein an at least one first peptide moiety has an amino acidsequence as defined above and the at least one second peptide moiety isthe reverse sequence thereof, wherein the at least two peptide moietiesare fused directly or via a linker to each other.

The linker comprises preferably 1 to 10, preferably 1 to 8, morepreferably 1 to 5, even more preferably 1 to 3, amino acid residues.

According to a particularly preferred embodiment of the presentinvention the linker, being preferably part of the turn, comprises orconsists of proline and/or glycine, preferably proline.

The isolated peptide of the present invention or a pharmaceuticalpreparation comprising at least one of said isolated peptides can beused to treat cancer of solid and non-solid tumors, includingmetastases, whereby the cancer is preferably selected from the groupconsisting of melanoma, rhabdomyosarcoma, glioblastoma, colorectalcancer, breast cancer, lymphoma, prostate cancer, pancreatic cancer,renal cancer, ovarian cancer and lung cancer. Particularly preferredcancers to be treated with the peptides of the present invention areglioblastoma and melanoma, preferably malignant melanoma. The peptidesof the present invention are preferably administered to a patient inneed thereof in an amount of 100 μg/kg body weight to 100 mg/kg bodyweight, preferably 1 mg/kg body weight to 50 mg/kg body weight, morepreferably 5 mg/kg body weight to 15 mg/kg body weight, in particular 10mg/kg body weight. Furthermore the peptides of the present invention arepreferably administered daily (e.g. three times a day, twice a day oronce a day), every 2^(nd), every 3^(rd), every 4^(th) or every 5^(th)day.

In order to obtain a pharmaceutical composition with even betteranti-cancer or antitumor activity additional agents exhibiting similarproperties as the peptides according to the present invention are added.Of course it is also possible to add agents with activities other thanthe peptides according to the present invention. These substances may behelpful in increasing the bioavailability such as for example increasingthe stability of the peptides or their delivery.

Such compositions according to the present invention may preferablyfurther comprise a pharmaceutically acceptable excipient.

The pharmaceutical composition of the present invention may consist ofthe peptide of the present invention alone or may be in the form of acomposition comprising the peptide of the present invention and apharmaceutically acceptable carrier. The pharmaceutically acceptablecarrier which can be used is not limited particularly and includes anexcipient, a binder, a lubricant, a colorant, a disintegrant, a buffer,an isotonic agent, a preservative, an anesthetic, and the like which canbe used in a medical field.

The composition of the present invention can be administered, dependingon the cancer to be treated locally or systemically by injection(subcutaneous, intracutaneous, intravenous, intraperitoneal, etc.), eyedropping, instillation, percutaneous administration, oraladministration, inhalation, etc. The peptides of the present inventioncan also be directly injected into the tumor/cancer to be treated.

Also, the dosage form such as injectable preparations (solutions,suspensions, emulsions, solids to be dissolved when used, etc.),tablets, capsules, granules, powders, liquids, liposome inclusions,ointments, gels, external powders, sprays, inhalating powders, eyedrops, eye ointment, suppositories, pessaries, and the like can beappropriately selected depending on the administration method, and thecomposition of the present invention can be accordingly formulated.

Another aspect of the present invention relates to the use of a peptideas defined above for the manufacturing of a medicament for treatingcancer in a mammal, in particular in a human patient.

A further aspect of the present invention relates to a method fortreating a mammal, in particular a human patient, suffering from cancerby administering to said mammal an effective amount of a peptide asdefined above.

As used herein, the term “therapeutically effective amount” or“effective amount” means that to a mammal an amount of the peptide ofthe present invention is administered which allows the reduction of thetumor cells within the body of at least 10%, preferably at least 20%,more preferably at least 50%, and more preferably sufficient to reduceby 90%. Generally, the dosage will vary with age, condition and sex, andcan be determined by one skilled in the art. The dosage can be adjustedby the individual physician in the event of any contraindications. Inany event, the effectiveness of treatment can be determined bymonitoring the presence of the cancer cells within the body.

According to a particularly preferred embodiment of the presentinvention the peptide of the present invention has/comprises/consists ofa sequence selected from the group consisting of SEQ ID. No. 125, SEQID. No. 127, SEQ ID. No. 128, SEQ ID. No. 106 and SEQ ID. No. 141 and isused in the treatment of glioblastoma and melanoma, preferably malignantmelanoma. The peptide of the present invention can be administered bydirectly injecting the peptide into the tumor/cancer.

1. An isolated peptide to be used in the treatment of cancer consistingof 12 to 50 amino acid residues comprising

-   -   at least two beta-strands, or    -   at least two alpha-helices or    -   at least one beta-strand and at least one alpha-helix,        wherein said beta-strands and/or alpha-helices are preferably        separated from each other by at least one turn, wherein the        peptide has a net positive charge of +7 or more.

2. Peptide for use according to embodiment 1, wherein the peptidecomprises at least 5 hydrophobic amino acid residues.

3. Peptide for use according to embodiment 1 or 2, wherein the isolatedpeptide comprises at least one peptide moiety having amino acid sequence(X₁)_(M)-X₂-(X₃)_(P)-X₄-(X₅)_(Q)-X₆-(X₇)_(S) or the reverse sequencethereof, wherein

-   -   X₁ is a hydrophobic amino acid, preferably selected from the        group consisting of phenylalanine (Phe), alanine (Ala), leucine        (Leu) and valine (Val),    -   X₂ is a hydrophobic amino acid, preferably tryptophan (Trp),    -   X₃ is selected from the group consisting of alanine (Ala),        arginine (Arg), glutamine (Gln), asparagine (Asn), proline        (Pro), isoleucine (Ile), leucine (Leu) and valine (Val),    -   X₄ is selected from the group consisting of isoleucine (Ile),        phenylalanine (Phe), tryptophan (Trp) and tyrosine (Tyr),    -   X₅ is selected from the group consisting of arginine (Arg),        lysine (Lys), tyrosine (Tyr) and phenylalanine (Phe),    -   X₆ is a hydrophobic amino acid, preferably selected from the        group consisting of isoleucine (Ile), tryptophan (Trp), valine        (Val) and leucine (Leu), and    -   X₇ is selected from the group consisting of arginine (Arg),        lysine (Lys), isoleucine (Ile) and serine (Ser), and wherein    -   M is 1 or 2,    -   Q is 1 or 2,    -   P is 2 or 3, and    -   S is 1, 2, 3 or 4 under the proviso that if (X₅)_(Q) is Arg-Arg        S is 1.

4. Peptide for use according to embodiment 3, wherein the at least onepeptide moiety has an amino acid sequence selected from the groupconsisting of FWQRIRKVR (SEQ ID No. 1), FWQRRIRKVRR (SEQ ID No. 2),FWQRKIRKVRK (SEQ ID No. 3), FWQRNIRIRR (SEQ ID No. 4), FWQRNIRKVR (SEQID No. 5), FWQRNIRVR (SEQ ID No. 6), FWQRNIRKVRR (SEQ ID No. 7),FWQRNIRKVKK (SEQ ID No. 8), FWQRNIRKVRRR (SEQ ID No. 9), FWQRNIRKVKKK(SEQ ID No. 10), FWQRNIRKVRRRR (SEQ ID No. 11), FWQRNIRKVRRRI (SEQ IDNo. 12), FWQRNIRKVKKKK (SEQ ID No. 13), FWQRNIRKVKKKI (SEQ ID No. 14),FWQRNIRKIR (SEQ ID No. 15), FWQRNIRKLR (SEQ ID No. 16), FWQRNIRKWR (SEQID No. 17), FWQRNWRKVR (SEQ ID No. 18), FWQRNFRKVR (SEQ ID No. 19),FWQRNYRKVR (SEQ ID No. 20), FWQRNIRKVS (SEQ ID No. 21), FWQRRIRIRR (SEQID No. 22), FWQRPIRKVR (SEQ ID No. 23), FWQRRIRKWR (SEQ ID No. 24),FWPRNIRKVR (SEQ ID No. 26), FWARNIRKVR (SEQ ID No. 27), FWIRNIRKVR (SEQID No. 28), FWLRNIRKVR (SEQ ID No. 29), FWVRNIRKVR (SEQ ID No. 30),FWQRNIFKVR (SEQ ID No. 31), FWQRNIYKVR (SEQ ID No. 32), FAWQRNIRKVR (SEQID No. 33), FLWQRNIRKVR (SEQ ID No. 35) and FVWQRNIRKVR (SEQ ID No. 36)or the reverse sequence thereof.

5. Peptide for use according to any one of embodiments 1 to 4, whereinthe isolated peptide comprises at least one peptide moiety having aminoacid sequence(X_(1′))_(M′)-X_(2′)-(X_(3′))_(P)-(X_(4′))_(Q′)-(X_(5′))_(T′)-(X_(6′))_(R′)-(X_(7′))_(S′)or the reverse sequence thereof, wherein

-   -   X_(1′) is a hydrophobic amino acid, preferably selected from the        group consisting of phenylalanine (Phe) and isoleucine (Ile),    -   X_(2′) is a hydrophobic amino acid, preferably tryptophan (Trp),    -   X_(3′) is selected from the group consisting of glycine (Gly),        asparagine (Asn), isoleucine (Ile) and phenylalanin (Phe),    -   X_(4′) is isoleucine (Ile) or tryptophan (Trp),    -   X_(5′) is arginine (Arg) or lysine (Lys),    -   X_(6′) is a hydrophobic amino acid, preferably selected from the        group consisting of isoleucine (Ile), tryptophan (Trp) and        valine (Val) and    -   X_(7′) is arginine (Arg), and wherein    -   M′ is 1 or 2,    -   T′ is 1 or 2,    -   R′ is 0 or 1,    -   P′ is 1, 2 or 3,    -   Q′ is 1, and    -   S′ is 0, 1 or 2.

6. Peptide for use according to embodiment 5, wherein the at least onepeptide moiety has an amino acid sequence selected from the groupconsisting of FWRIRKWR (SEQ ID No. 37), FWRIRKVR (SEQ ID No. 38), FWRWRR(SEQ ID No. 39), FWRRWRR (SEQ ID No. 40), FWRRWIRR (SEQ ID No. 41),FWRGWRIRR (SEQ ID No. 42), FWRRFWRR (SEQ ID No. 43), FWRWRWR (SEQ ID No.44), FWRIWRWR (SEQ ID No. 45), FWRIWRIWR (SEQ ID No. 46), FWRNIRKWR (SEQID No. 47) and FWRRRIRIRR (SEQ ID No. 48) or the reverse sequencethereof.

7. Peptide for use according to any one of embodiments 1 to 6, whereinthe isolated peptide comprises at least one peptide moiety having aminoacid sequence(X_(1″))_(M′)X_(2″)-(X_(3″))_(P′)-(X_(4″))_(Q″)-(X_(5″))_(R″)-(X_(6″))_(S″)or the reverse sequence thereof, wherein

-   -   X_(1″) is a hydrophobic amino acid, preferably selected from the        group consisting of proline (Pro) and phenylalanine (Phe),    -   X_(2″) is a hydrophobic amino acid, preferably tryptophan (Trp),    -   X_(3″) is selected from the group consisting of alanine (Ala),        arginine (Arg), glutamine (Gln), lysine (Lys), tryptophan (Trp)        and isoleucine (Ile),    -   X_(4″) is selected from the group consisting of arginine (Arg)        and aspartate (Asp),    -   X_(5″) is a hydrophobic amino acid, preferably selected from the        group consisting of isoleucine (Ile), tryptophan (Trp),        phenylalanine (Phe), valine (Val) and leucine (Leu), and    -   X_(6″) is selected from the group consisting of arginine (Arg),        lysine (Lys), isoleucine (Ile), serine (Ser) and aspartate        (Asp), and wherein    -   M″ is 0, 1, 2 or 3,    -   Q″ is 0, 1, 2 or 3,    -   R″ is 1 or 2,    -   P″ is 1, 2 or 3, and    -   S″ is 1, 2 or 3.

8. Peptide for use according to embodiment 7, wherein the at least onepeptide moiety has an amino acid sequence selected from the groupconsisting of PFWRWRIWR (SEQ ID No. 50), PFWRIRIRR (SEQ ID No. 51),PFWRQRIRR (SEQ ID No. 52), PFWRARIRR (SEQ ID No. 53), PFWRKRIRR (SEQ IDNo. 54), PFWRKRLRR (SEQ ID No. 55), PFWRKRWRR (SEQ ID No. 56), PFWRRRIRR(SEQ ID No. 57), PFWRRRWRR (SEQ ID No. 58), PFWRIRIRRD (SEQ ID No. 59),PFFWRIRIRR (SEQ ID No. 60), PWRIRIRR (SEQ ID No. 61), PFWRRQIRR (SEQ IDNo. 81), PFWRKKLKR (SEQ ID No. 82), PWRRIRR (SEQ ID No. 83), PWRRKIRR(SEQ ID No. 84) and PFWRRIRIRR (SEQ ID No. 85) or the reverse sequencethereof.

9. Peptide for use according to any one of embodiments 1 to 8, whereinthe isolated peptide comprises at least one peptide moiety having aminoacid sequence(X_(1′″))_(M′″)-(X_(2′″))_(O′″)-X_(3′″)-(X_(4′″))_(P′″)-(X_(5′″))_(Q′″)-(X_(6′″))_(T′″)-(X_(7′″))_(R′″)-(X_(8′″))_(S′″)or the reverse sequence thereof, wherein

-   -   X_(1′″) is a hydrophobic amino acid, preferably selected from        the group consisting of proline (Pro) and phenylalanine (Phe),    -   X_(2′″) is a basic amino acid, preferably arginine (Arg),    -   X_(3′″) is a hydrophobic amino acid, preferably tryptophan        (Trp),    -   X_(4′″) is selected from the group consisting of alanine (Ala),        arginine (Arg), glutamine (Gln), asparagine (Asn) and lysine        (Lys),    -   X_(5′″) is selected from the group consisting of isoleucine        (Ile), phenylalanine (Phe) and tryptophan (Trp),    -   X_(6′″) is selected from the group consisting of glutamine        (Gln), arginine (Arg) and asparagine (Asn),    -   X_(7′″) is a hydrophobic amino acid, preferably selected from        the group consisting of isoleucine (Ile), tryptophan (Trp) and        phenylalanine (Phe), and    -   X_(8′″) is arginine (Arg), and wherein    -   M′″ is 0, 1, 2 or 3,    -   T′″ is 0, 1, 2 or 3,    -   O′″ is 0 or 1,    -   P′″ is 1, 2 or 3,    -   Q′″ is 1 or 2, and    -   R′″ and S′″ are 0, 1 or 2.

10. Peptide for use according to embodiment 9, wherein the at least onepeptide moiety has an amino acid sequence selected from the groupconsisting of FWRNIRIRR (SEQ ID No. 72), FWQRIRIRR (SEQ ID No. 73),FWRWRIWR (SEQ ID No. 74), FWRIRIRR (SEQ ID No. 75), FWRNIRIWRR (SEQ IDNo. 76) and FWRNIRIRR (SEQ ID No. 77) or the reverse sequence thereof.

11. Peptide for use according to any one of embodiments 1 to 10, whereinthe isolated peptide comprises at least one peptide moiety having anamino acid sequence selected from the group consisting of RFWQRNIRKVRR(SEQ ID No. 62), RFWQRNIRKYR (SEQ ID No. 63), PFWQRNIRKWR (SEQ ID No.64), RFRWQRNIRKYRR (SEQ ID No. 65), RWKRINRQWF (SEQ ID No. 66), KRFCFKK(SEQ ID No. 67), KRFSFKKC (SEQ ID No. 68), KRWSWKK (SEQ ID No. 69),FRFSFKK (SEQ ID No. 70), RRFWFRR (SEQ ID No. 71), RFWQRNIRIRR (SEQ IDNo. 78), RWQRNIRIRR (SEQ ID No. 79) and RRWFWRR (SEQ ID No. 86) or thereverse sequence thereof.

12. Peptide for use according to any one of embodiments 1 to 11, whereinthe isolated peptide comprises at least one peptide moiety having anamino acid sequence selected from the group consisting of FIWQRNIRKVR(SEQ ID No. 34), FIWRWRWR (SEQ ID No. 49) and RRIRINRQWF (SEQ ID No. 80)or the reverse sequence thereof.

13. Peptide for use according to any one of embodiments 1 to 12, whereinat least two peptide moieties having an amino acid sequence as definedin any one of embodiments 4 to 13 or the reverse sequence thereof arefused directly or via a linker to each other.

14. Peptide for use according to any one of embodiments 1 to 12, whereinsaid isolated peptide comprises at least two, preferably two, peptidemoieties having the same amino acid sequence and being selected from thepeptide moieties having an amino acid sequence as defined in any one ofembodiments 4 to 13 or the reverse sequence thereof, wherein the atleast two peptide moieties are fused directly or via a linker to eachother.

15. Peptide for use according to any one of embodiments 1 to 12, whereinsaid isolated peptide comprises at least two, preferably two, peptidemoieties, wherein an at least one first peptide moiety has an amino acidsequence as defined in any one of embodiments 4 to 13 and the at leastone second peptide moiety is the reverse sequence thereof, wherein theat least two peptide moieties are fused directly or via a linker to eachother.

16. Peptide for use according to any one of embodiments 13 to 15,wherein the linker comprises 1 to 10, preferably 1 to 8, more preferably1 to 5, even more preferably 1 to 3, amino acid residues.

17. Peptide for use according to any one of embodiments 13 to 16,wherein the linker comprises proline and/or glycine residues.

18. Peptide for use according to any one of embodiments 13 to 17,wherein the linker consists of one, two or three, preferably one,proline residue.

19. Peptide for use according to any one of embodiments 1 to 18, whereinthe cancer is selected from solid and non-solid tumors, includingmetastases.

20. Peptide for use according to any one of embodiments 1 to 19, whereinthe cancer is selected from the group consisting of melanoma,rhabdomyosarcoma, glioblastoma, colorectal cancer, breast cancer,lymphoma, prostate cancer, pancreatic cancer, renal cancer, ovariancancer and lung cancer.

21. Use of a peptide as defined in any one of embodiments 1 to 18 forthe manufacturing of a medicament for treating cancer in a mammal, inparticular in a human patient.

22. Method for treating a mammal, in particular a human patient,suffering from cancer by administering to said mammal an effectiveamount of a peptide as defined in any one of embodiments 1 to 18.

DESCRIPTION OF FIGURES

The present invention is further illustrated by the following figuresand examples, however, without being restricted thereto.

FIG. 1 shows the secondary structures of PEP-322 (A) and R-DIM-P-PEP-322(B) in Hepes buffer (first bar) or presence of SDS and DPC at peptide tosurfactant ratios of 1:25 and 1:100, determined using CD spectroscopy.Bottom shows α-helical content in dark gray; second from bottom showsβ-sheet in light grey; third from bottom shows turns in middle grey;random coil structures are shown in dark grey at the top.

FIG. 2 shows the secondary structures of DIM-PEP-318 (A) andR-DIM-P-PEP-322 (B) in the absence and presence of SDS and DPC atpeptide to surfactant ratios of 1:25 and 1:100, determined using CDspectroscopy. Bottom shows α-helical content in dark gray; second frombottom shows β-sheet in light grey; third from bottom shows turns inmiddle grey; random coil structures are shown in dark grey at the top.

FIG. 3 shows peptide toxicity-PI-uptake of cancer and non-cancer celllines: (A) Concentration dependent cytotoxic activity of PEP-322 (●) andR-DIM-P-PEP-322 (▾) against melanoma cell line SBcl-2 after 8 hours ofincubation with peptides; (B) Concentration dependent cytotoxic activityagainst primary cultures of differentiated non-tumorigenic melanocytesafter 8 hour of incubation with peptides; (C) specificity of peptides at20 μM peptide concentration after 8 h of incubation displayed asPI-uptake ratio of SBcl-2 vs. melanocytes and WM164 vs. melanocytes.

FIG. 4 shows PI-uptake of various cancerous and non-cancerous cell linesupon incubation with 20 μM peptide: Time dependent cytotoxic activity ofPEP-322 (●), R-DIM-P-PEP-322 (▾), PEP-318 (Δ) and DIM-PEP-318 (▪)against melanoma cell line SBcl-2 (A), melanoma metastasis WM164 (B),Rhabdomyosarcoma cell line TE671 (C), differentiated non-tumorigenicmelanocytes cell lines (D) and normal human dermal fibroblast cell lineNHDF (E) at 20 μM peptide concentration is shown.

FIG. 5 shows cytotoxicity of peptides after 8 h of incubation againstSBcl-2 melanoma cell line, WM164 melanoma metastasis, TE671rhabdomyosarcoma cell line, differentiated non-tumorigenic melanocytecell line and NHDF normal human dermal fibroblast cell line.

FIG. 6 shows cytotoxic activity of PEP-322 (A), R-DIM-P-PEP-322 (B) andDIM-PEP-318 (C) determined by MTS cell proliferation assay againstmelanoma cell line SBcl-2 melanoma metastasis WM164 and non-cancer humandermal fibroblasts (NHDF). Cells were kept in the appropriate mediumduring incubation time of 24 h.

FIG. 7 shows spectrofluorimetric analysis of Caspase 3/7-activity ofmelanoma cell line SBcl-2 upon incubation of 4 h with differentconcentrations (5 μM, 10 μM, 20 μM, 40 μM, 80 μM) of peptideR-DIM-P-PEP-322 (black) and DIM-PEP-318 (white).

FIGS. 8 to 11 show cytotoxicity of peptides after 1 h, 2 h, 4 h and 8 hof incubation, respectively, against SBcl-2 melanoma cell line, WM164melanoma metastasis, TE671 rhabdomyosarcoma cell line, differentiatednon-tumorigenic melanocyte cell line and NHDF normal human dermalfibroblast cell line.

FIG. 12 shows cytotoxicity of specific peptides R-DIM-P-PEP-322 andR-DIM-PEP-316 and less specific peptides R-DIM-PEP-337 and DIM-PEP-318and weakly active human Lactoferricin (hLFcin)(37-61), which containstwo cysteins (Cys3 and Cys20) connected via a disulfide bond, after 8 hof incubation, respectively, against U87 mg glioblastoma cell line, A375melanoma cell line and NHDF-c normal human dermal fibroblast cell lineand PEP-FOLD secondary structure predictions of peptides.

FIG. 13 shows secondary structure predictions of the peptides PEP-322(A), R-DIM-P-PEP-322 (B), R-DIM-P-PEP-334 (C), R-DIM-PEP-316 (D),R-DIM-PEP-337 (E), DIM-PEP-324 (F) and DIM-318 (G), analyzed by theprogram PEP-FOLD.

FIG. 14 shows cytotoxicity of peptides R-DIM-P-PEP-316, DIM-PEP-317,DIM-PEP-318, DIM-PEP-322, R-DIM-PEP-322, R-DIM-P-PEP-322, R-DIM-PEP-323,DIM-PEP-324, R-DIM-P-PEP-324, R-DIM-P-PEP-330, R-DIM-P-PEP-332,R-DIM-P-PEP-334 and R-DIM-PEP-337 and human Lactoferricin(hLFcin)(37-61) after 1 h, 2 h, 4 h and 8 h of incubation, respectively,against A375 melanoma cell line.

FIG. 15 shows cytotoxicity of peptides R-DIM-P-PEP-316, DIM-PEP-317,DIM-PEP-318, DIM-PEP-322, R-DIM-PEP-322, R-DIM-P-PEP-322, R-DIM-PEP-323,DIM-PEP-324, R-DIM-P-PEP-324, R-DIM-P-PEP-330, R-DIM-P-PEP-332,R-DIM-P-PEP-334 and R-DIM-PEP-337 and human Lactoferricin(hLFcin)(37-61) after 1 h, 2 h, 4 h and 8 h of incubation, respectively,against U87 mg glioblastoma cell line.

EXAMPLES Experimental Procedures

Materials and Peptide Synthesis

1,2-Dihexadecanoyl-sn-glycero-3-phosphocholine (DPPC),1-Hexadecanoyl-2-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (POPC),1,2-Dihexadecanoyl-sn-glycero-3-phospho-L-serine (Na-salt) (DPPS) and1-Hexadecanoyl-2-(9Z-octadecenoyl)-sn-glycero-3-phospho-L-serine(Na-salt) (POPS) were purchased from Avanti Polar Lipids, Inc. (USA),and used without further purification. Stock solutions of DPPC and POPCwere prepared in CHCl₃/CH₃OH (2:1, v/v), stock solutions of DPPS andPOPS were prepared in CHCl₃/CH₃OH (9:1, v/v) and stored at −18° C.

The amidated peptides PEP-322 (PFWRIRIRR-NH₂, M=1298.6 g/mole,DIM-PEP-322 (2580.2), M=g/mole, R-DIM-PEP-322 (2580.2), M=g/mole,R-DIM-P-PEP-322 (PFWRIRIRRPRRIRIRWFP-NH₂, M=2677.4 g/mole), PEP-318(FWQRRIRRWRR-NH₂, M=1715.0 g/mol), DIM-PEP-318(FWQRRIRRWRRFWQRRIRRWRR-NH₂, M=3413.1 g/mol), PEP-324 (PFFWRIRIRR-NH₂,M=1445.8 g/mol), DIM-PEP-324 (PFFWRIRIRRPFFWRIRIRR-NH₂, M=2874.6 g/mol),PEP-316 (RWKRINRQWF-NH₂, M=1488.8 g/mol) and R-DIM-PEP-316(RWKRINRQWFFWQRNIRKWR-NH₂, M=2960.5 g/mol) were purchased from NeoMPS,Inc. (San Diego, Calif., USA). Human lactoferricin (hLFcin) (37-61)(TKCFQWQRNMRKVRGPPVSCIKRDS (SEQ ID No. 207), M=3020.5 g/mol) waspurchased from Anaspec, Inc (Fremont, Calif., USA). The puritieswere >95% as determined by RP-HPLC. Peptides were dissolved in aceticacid (0.1%, v/v) at a concentration of 3 mg/ml. Peptide solutions werestored at 4° C. and concentrations were determined photometrically at280 nm.

ANTS (8-aminonaphthalene-1,3,6-trisulfonic acid, disodium salt) and DPX(p-xylene-bis-pyridinium bromide) used for permeability studies werepurchased from Molecular Probes (Eugene, Oreg.).

The peptides of the present invention, which have been used in part inthe present example are indicated in Table 1:

TABLE 1 List of sequences of peptide moieties and isolated peptides(-retro); the present invention includes all sequences inisolated peptide (The term ″DIM-″ used hereinafter was usedto assign a ″di-moiety″ peptide according to the presentinvention) (peptide moietysequence + peptide moietysequence)isolated peptide retro (R-DIM-) (peptide moiety sequence +peptide moiety retro sequence), isolated peptide with linker (DIM-X-) (peptide moiety sequence + X + peptide moietysequence) isolated peptide retro with linker X (R-DIM-X-)(peptide moiety sequence + X + peptide moiety retro sequence)  (X =(Pro and/or, preferably or, Gly)1.3, whereby X ispreferably a single proline residue); the present inventionincludes as well trimer and tetrameric derivatives termed aspolymeric versions of the isolated peptide according tothe present invention, although the more consequent wordingwould be ″poly-moietic peptide″ (″tri-moiety peptide″, ″tetra-moiety peptide″, ″penta-moiety peptide″, etc.); - represents a peptide bond between the peptide moieties andbetween the peptides and the linker; ″retro sequence″ or″reverse sequence″ refers to an amino acid sequence comprisinga sequence which has a reverse order than the amino acidsequence from which it is derived from (e.g. the retrosequence of ABCDE is EDCBA). Designation Sequence SEQ ID No.PEP (parent) FQWQRNIRKVR  87 DIM-PEP FQWQRNIRKVR-FQWQRNIRKVR  88DIM-X-PEP FQWQRNIRKVR X FQWQRNIRKVR  89 R-DIM-PEPFQWQRNIRKVR RVKRINRQWQF  90 R-DIM-X-PEP FQWQRNIRKVR X RVKRINRQWQF  91PEP-313 FWQRNIRIRR   4 DIM-PEP-313 FWQRNIRIRR FWQRNIRIRR  92DIM-X-PEP-313 FWQRNIRIRR X FWQRNIRIRR  93 R-DIM-PEP-313FWQRNIRIRR RRIRINRQWF  94 R-DIM-X-PEP-313 FWQRNIRIRR X RRIRINRQWF  95PEP-314 RRIRINRQWF  80 DIM-PEP-314 RRIRINRQWF RRIRINRQWF  96DIM-X-PEP-314 RRIRINRQWF X RRIRINRQWF  97 R-DIM-PEP-314RRIRINRQWF FWQRNIRIRR  98 R-DIM-X-PEP-314 RRIRINRQWF X FWQRNIRIRR  99PEP-315 FWQRNIRKWR  17 DIM-PEP-315 FWQRNIRKWR FWQRNIRKWR 100DIM-X-PEP-315 FWQRNIRKWR X FWQRNIRKWR 101 R-DIM-PEP-315FWQRNIRKWR RWKRINRQWF 102 R-DIM-X-PEP-315 FWQRNIRKWR X RWKRINRQWF 103PEP-316 RWKRINRQWF  66 DIM-PEP-316 RWKRINRQWF RWKRINRQWF 104DIM-X-PEP-316 RWKRINRQWF X RWKRINRQWF 105 R-DIM-PEP-316RWKRINRQWF FWQRNIRKWR 106 R-DIM-X-PEP-316 RWKRINRQWF X FWQRNIRKWR 107PEP-317 FWQRRIRKWR  24 DIM-PEP-317 FWQRRIRKWR FWQRRIRKWR 108DIM-X-PEP-317 FWQRRIRKWR X FWQRRIRKWR 109 R-DIM-PEP-317FWQRRIRKWR RWKRIRRQWF 110 R-DIM-X-PEP-317 FWQRRIRKWR X RWKRIRRQWF 111PEP-318 FWQRRIRRWRR 112 DIM-PEP-318 FWQRRIRRWRR FWQRRIRRWRR 113DIM-X-PEP-318 FWQRRIRRWRR X FWQRRIRRWRR 114 R-DIM-PEP-318FWQRRIRRWRR RRWRRIRRQWF 115 R-DIM-X-PEP-318 FWQRRIRRWRR X RRWRRIRRQWF116 PEP-319 PFWQRNIRKWR  64 DIM-PEP-319 PFWQRNIRKWR PFWQRNIRKWR 117DIM-X-PEP-319 PFWQRNIRKWR X PFWQRNIRKWR 118 R-DIM-PEP-319PFWQRNIRKWR RWKRINRQWFP 119 R-DIM-X-PEP-319 PFWQRNIRKWR X RWKRINRQWFP120 PEP-320 FWRNIRKWR  47 DIM-PEP-320 FWRNIRKWR FWRNIRKWR 121DIM-X-PEP-320 FWRNIRKWR X FWRNIRKWR 122 R-DIM-PEP-320FWRNIRKWR RWKRINRWF 123 R-DIM-X-PEP-320 FWRNIRKWR X RWKRINRWF 124PEP-322 PFWRIRIRR  51 DIM-PEP-322 PFWRIRIRR PFWRIRIRR 125 DIM-X-PEP-322PFWRIRIRR X PFWRIRIRR 126 R-DIM-PEP-322 PFWRIRIRR RRIRIRWFP 127R-DIM-X-PEP-322 PFWRIRIRR X RRIRIRWFP 128 PEP-215 FWRIRIRR  75DIM-PEP-215 FWRIRIRR FWRIRIRR 129 DIM-X-PEP-215 FWRIRIRR X FWRIRIRR 130R-DIM-PEP-215 FWRIRIRR RRIRIRWF 131 R-DIM-X-PEP-215 FWRIRIRR X RRIRIRWF132 PEP-227 FWRRFWRR  43 DIM-PEP-227 FWRRFWRR FWRRFWRR 133 DIM-X-PEP-227FWRRFWRR X FWRRFWRR 134 R-DIM-PEP-227 FWRRFWRR RRWFRRWF 135R-DIM-X-PEP-227 FWRRFWRR X RRWFRRWF 136 PEP-323 PFWRIRIRRD  59DIM-PEP-323 PFWRIRIRRD PFWRIRIRRD 137 DIM-X-PEP -323PFWRIRIRRD X PFWRIRIRRD 138 R-DIM-PEP -323 PFWRIRIRRD DRRIRIRWFP 139R-DIM-X-PEP-323 PFWRIRIRRD X DRRIRIRWFP 140 PEP-324 PFFWRIRIRR  60DIM-PEP-324 PFFWRIRIRR PFFWRIRIRR 141 DIM-X-PEP -324PFFWRIRIRR X PFFWRIRIRR 142 R-DIM-PEP-324 PFFWRIRIRR RRIRIRWFFP 143R-DIM-X-PEP-324 PFFWRIRIRR X RRIRIRWFFP 144 PEP-325 PFWRQRIRR  52DIM-PEP-325 PFWRQRIRR PFWRQRIRR 145 DIM-X-PEP -325 PFWRQRIRR X PFWRQRIRR146 R-DIM-PEP -325 PFWRQRIRR RRIRQRWFP 147 R-DIM-X-PEP-325PFWRQRIRR X RRIRQRWFP 148 PEP-326 PFWRRQIRR  81 DIM-PEP-326PFWRRQIRR PFWRRQIRR 149 DIM-X-PEP -326 PFWRRQIRR X PFWRRQIRR 150R-DIM-PEP -326 PFWRRQIRR RRIQRRWFP 151 R-DIM-X-PEP-326PFWRRQIRR X RRIQRRWFP 152 PEP-327 PFWRARIRR  53 DIM-PEP-327PFWRARIRR PFWRARIRR 153 DIM-X-PEP -327 PFWRARIRR X PFWRARIRR 154R-DIM-PEP -327 PFWRARIRR RRIRARWFP 155 R-DIM-X-PEP-327PFWRARIRR X RRIRARWFP 156 PEP-328 PFWRKRIRR  54 DIM-PEP-328PFWRKRIRR PFWRKRIRR 157 DIM-X-PEP -328 PFWRKRIRR X PFWRKRIRR 158R-DIM-PEP -328 PFWRKRIRR RRIRKRWFP 159 R-DIM-X-PEP-328PFWRKRIRR X RRIRKRWFP 160 PEP-329 PFWRKRLRR  55 DIM-PEP-329PFWRKRLRR PFWRKRLRR 161 DIM-X-PEP -329 PFWRKRLRR X PFWRKRLRR 162R-DIM-PEP -329 PFWRKRLRR RRLRKRWFP 163 R-DIM-X-PEP-329PFWRKRLRR X RRLRKRWFP 164 PEP-330 PFWRKKLKR  82 DIM-PEP-330PFWRKKLKR PFWRKKLKR 165 DIM-X-PEP -330 PFWRKKLKR X PFWRKKLKR 166R-DIM-PEP -330 PFWRKKLKR RKLKKRWFP 167 R-DIM-X-PEP-330PFWRKKLKR X RKLKKRWFP 168 PEP-331 PFWRKRWRR  56 DIM-PEP-331PFWRKRWRR PFWRKRWRR 169 DIM-X-PEP-331 PFWRKRWRR X PFWRKRWRR 170R-DIM-PEP-331 PFWRKRWRR RRWRKRWFP 171 R-DIM-X-PEP-331PFWRKRWRR X RRWRKRWFP 172 PEP-332 PFWRRRIRR  57 DIM-PEP-332PFWRRRIRR PFWRRRIRR 173 DIM-X-PEP -332 PFWRRRIRR X PFWRRRIRR 174R-DIM-PEP -332 PFWRRRIRR RRIRRRWFP 175 R-DIM-X-PEP-332PFWRRRIRR X RRIRRRWFP 176 PEP-333 PFWRRRWRR  58 DIM-PEP-333PFWRRRWRR PFWRRRWRR 177 DIM-X-PEP -333 PFWRRRWRR X PFWRRRWRR 178R-DIM-PEP-333 PFWRRRWRR RRWRRRWFP 179 R-DIM-X-PEP -333PFWRRRWRR X RRWRRRWFP 180 PEP-334 PWRIRIRR  61 DIM-PEP-334PWRIRIRR PWRIRIRR 181 DIM-X-PEP-334 PWRIRIRR X PWRIRIRR 182R-DIM-PEP -334 PWRIRIRR RRIRIRWP 183 R-DIM-X-PEP-334 PWRIRIRR X RRIRIRWP184 PEP-335 PWRRIRR  83 DIM-PEP-335 PWRRIRR PWRRIRR 185 DIM-X-PEP -335PWRRIRR X PWRRIRR 186 R-DIM-PEP -335 PWRRIRR RRIRRWP 187R-DIM-X-PEP -335 PWRRIRR X RRIRRWP 188 PEP-336 PWRRKIRR  84 DIM-PEP-336PWRRKIRR PWRRKIRR 189 DIM-X-PEP-336 PWRRKIRR X PWRRKIRR 190R-DIM-PEP -336 PWRRKIRR RRIKRRWP 191 R-DIM-X-PEP -336PWRRKIRR X RRIKRRWP 192 PEP-337 PFWRRRIRIRR 193 DIM-PEP-337PFWRRRIRIRR PFWRRRIRIRR 194 DIM-X-PEP-337 PFWRRRIRIRR X PFWRRRIRIRR 195R-DIM-PEP-337 PFWRRRIRIRR RRIRIRRRWFP 196 R-DIM-X-PEP-337RRIRIRRRWFP X RRIRIRRRWFP 197 PEP-338 RRWFFWRR 198 DIM-PEP-338RRWFFWRR RRWFFWRR 199 DIM-X-PEP-338 RRWFFWRR X RRWFFWRR 200R-DIM-PEP-338 RRWFFWRR RRWFFWRR 199 R-DIM-X-PEP-338 RRWFFWRR X RRWFFWRR200 PEP-339 RRWFWRR  86 DIM-PEP-339 RRWFWRR RRWFWRR 201 DIM-X-PEP-339RRWFWRR X RRWFWRR 202 R-DIM-PEP-339 RRWFWRR RRWFWRR 201 R-DIM-X-PEP-339RRWFWRR X RRWFWRR 202

Preparation of Liposomes

Appropriate amounts of respective phospholipid stock solution were driedunder a stream of nitrogen and stored in vacuum overnight to completelyremove organic solvents. The dry lipid film was then dispersed inphosphate buffered saline (PBS, 20 mM NaPi, 130 mM NaCl, pH 7.4) andhydrated at a temperature well above the gel to fluid phase transitionof the respective phospholipid under intermittent vigorousvortex-mixing. The lipid concentration was 0.1 weight % for calorimetricexperiments. Hydration was carried out in presence or absence ofpeptides at a lipid to peptide ratio of 25:1 and 12.5:1 using a protocoldescribed for POPS (Jimenez-Monreal, A. M. et al. Biochim Biophys Acta1373(1998), 209-219), DPPS (Jing, W. et al. J Peptide Sci 11(2005),735-743) and DPPC (Sevcsik, E. et al. Biochim Biophys Acta 1768 (2007)2586-2596). The fully hydrated samples were stored at room temperatureuntil measurement.

Differential Scanning Calorimetry (DSC)

DSC experiments were performed with a differential scanning calorimeter(VP-DSC) from MicroCal, Inc. (USA). Heating scans were performed at ascan rate of 30° C./h (pre-scan thermostating 30 min) with a finaltemperature of approximately 10° C. above the main transitiontemperature (T_(m)) and cooling scans at the same scan rate (pre-scanthermostating 1 min) with a final temperature approximately 20° C. belowT_(m) The heating/cooling cycle was performed three times. Enthalpieswere calculated by integration of the peak areas after normalization tophospholipid concentration and baseline adjustment using the MicroCalOrigin software (VP-DSC version). The phase transition temperature wasdefined as the temperature at the peak maximum (McElhaney, R. N. ChemPhys Lipids 30(1982), 229-259).

Circular Dichroism Spectroscopy

Measurements were performed on a Jasco J 715 Spectropolarimeter (Jasco,Germany) at room temperature using quartz cuvettes with an optical pathlength of 0.02 cm. The CD spectra were measured between 260 nm and 180nm with a 0.2 nm step resolution. To improve accuracy 5 scans wereaveraged. Peptides were dissolved in 10 mM Hepes (pH 7.4) to a finalconcentration of 100 μM. Spectra were measured in the absence andpresence of 1 mM sodium dodecyl sulfate (SDS) and 1 mMdodecylphosphocholine (DPC) mimicking cancer and healthy mammalianmembranes, respectively. The respective peptide to surfactant molarratios were 1:25 and 1:100. Background signals were abstracted aftermeasurements. Percentage secondary structure calculations were doneusing Dichroweb, CDSSR Convolution Program using reference set 4(Whitmore, L. and Wallace, B. A. Biopolymers 89(2008), 392-400 andNucleic Acids Res. 32 (2004), W668-W673.

Fluorescence Spectroscopy

Fluorescence spectroscopy experiments were performed using a SPEX FluoroMax-3 spectrofluorimeter (Jobin-Yvon, France) and spectra were analyzedwith Datamax software.

ANTS/DPX Leakage

Leakage of aqueous contents from liposomes was determined using the8-aminonaphthalene-1,3,6-trisulfonic acid/p-xylene-bis-pyridiniumbromide (ANTS/DPX) assay. Lipid films were hydrated with 12.5 mM ANTS,45 mM DPX, 68 mM NaCl, 10 mM HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) at pH 7.4 followinga standard procedure.

Subsequently, the dispersions were extruded 20 times through apolycarbonate filter of 0.1 μm pore size to obtain LUVs. Unilamellarityand size were tested by X-ray and dynamic light scattering,respectively. The ANTS/DPX encapsulating vesicles were separated fromfree ANTS/DPX by exclusion chromatography using a column filled withSephadex G-75 (Amersham Biosciences) fine gel swollen in an iso-smoticbuffer (10 mM HEPES, 140 mM NaCl, pH 7.4). The void volume fractionswere collected and the phospholipid concentration was determined byphosphate analysis (Broekhuyse, R. M. Biochim. Biophys. Acta 152(2005),307-315; Tao, T. and Cho, J. Biochemistry 18(1979), 2759-2765).

The fluorescence measurements were performed in 2 mL of the isosmoticbuffer in a quartz cuvette at room temperature. Aliquots of LUVs werediluted with the iso-osmotic buffer to a final lipid concentration of 50μM. Fluorescence spectra were obtained at 37° C. using an excitationwavelength of 360 nm and an emission wavelength of 530 nm and a slitwidth of 5 nm for both excitation and emission monochromators.Fluorescence emission was recorded as a function of time before andafter the addition of incremental amounts of peptide. The fluorescenceincrease due to leakage and subsequent dilution of quenched dye wasmeasured after addition of peptides. Peptides were added to finalconcentrations of 2, 4 and 8 corresponding to peptide to lipid molarratios of 1:25, 1:12.5 and 1:6.25, respectively.

Data are presented in terms of fluorescence intensity (I_(F)):

$I_{F} = \frac{F - F_{0}}{F_{{ma}\; x} - F_{0}}$F is the measured fluorescence, F₀ the initial fluorescence withoutpeptide and F_(max) the fluorescence corresponding to 100% leakagegained by addition of 1% Triton X-100.

Tryptophan Quenching

Tryptophan fluorescence spectra were obtained at room temperature usingan excitation wavelength of 282 nm and a slit width of 5 nm forexcitation and emission monochromators. Quenching of Tryptophan wascarried out in the presence and absence of phospholipid liposomes (lipidto peptide ratio 25:1) using 0.1, 0.4 and 0.7 M acrylamide. The datawere analyzed according to the Stern-Volmer equation:F ₀ /F=1+K _(SV) [Q]where F₀ and F represent the fluorescence emission intensities in theabsence and presence of the quencher molecule (Q) and K_(SV) is theStern-Volmer quenching constant, which is a quantitative measure for theaccessibility of tryptophan to acrylamide (Tao, T. and Cho, J.Biochemistry 18(1979), 2759-2765).

Cell Lines and Culture

The primary human melanoma cell line SBcl2 and the metastatic melanomaWM164 were maintained in RPMI (Sigma) supplemented with 2% FBS, 2%L-glutamine and 1% Pen/Strep. Glioblastoma (U87-mg) purchased from CLS(Cell Line Service Heidelberg, Germany) and Rhabdomyosarcoma cell lines(TE671) purchased from ECAAC (Health Protection Agency CultureCollections Salisbury, UK) are cultured in Dulbecco's Modified EagleMedium (DMEM) with addition of 2 mM Glutamine, 10% FBS (fetal bovineserum). Melanoma cell line A375 CLS (Cell Line Service Heidelberg,Germany) was cultured in Dulbecco's Modified Eagle Medium (DMEM) (PAA)with addition of 2 mM Glutamine and 10% FBS (fetal bovine serum. Humanmelanocytes used as healthy control cells: were isolated from theforeskin). The foreskin was cut into small pieces and incubated with0.3% trypsin (PAA) overnight at 4° C. and for one hour at 37° C.Epidermis was separated. Cells were mechanically removed from the celllayer and centrifuged at 300 g for 3 min. The pellet was resuspended inmelanocyte growth media (Biomedica). Melanocytes were further culturedin human melanocytes growth medium (PromoCell GmbH). Normal human dermalfibroblasts (NHDF) purchased from (PromoCell GmbH) were cultured infibroblast growth medium 2 (PromoCell GmbH). All cells were kept in a 5%CO₂ atmosphere at 37° C. At 90% confluency cell-culture flasks werepassaged with accutase. All cell cultures were periodically checked formycoplasma.

PI-Uptake Assay

For detection of PI-uptake by fluorescence spectroscopy experiments wereperformed according to the following protocol.

Cells were collected, resuspended in media and diluted to aconcentration of 10⁶ cells/ml. Aliquots of 10⁵ cells were incubated withpeptides for up to 8 hours at 37° C. and 5% CO₂. PI was added and cellswere again incubated for 5 min at room temperature in the dark.Excitation and emission wavelengths were 536 nm and 617 nm,respectively.

Cytotoxicity was calculated from the percentage of PI positive cells inmedia alone (P₀) and in the presence of peptide (P_(X)). Triton-X wasused to determine 100% of PI positive cells (P₁₀₀)

${{\%\mspace{14mu}{PI}} - {uptake}} = \frac{100*\left( {P_{X} - P_{0}} \right)}{\left( {P_{100} - P_{0}} \right)}$For detection of PI-uptake by fluorescence microscopy experiments wereperformed on a Leica DMI6000 B with IMC in connection with a LeicaDFC360 FX camera and AF 6000 software.

Cells (1-5×10⁴) were seeded on Ibidi μ-Slide 8 wells and grown in 300 μlmedia for 2-3 days to a confluent layer. Propidium iodide (PI, 2 μl of50 μg/ml in PBS) was added to the well and cell status was checked after5 min of incubation in the dark at room temperature. Then, peptides wereadded to the desired concentration and peptide effect was followedimmediately. Pictures were taken every 5 or 15 min for up to 8 h fromthe same section of cells. Excitation and emission wavelength were asfollows: PI excitation, 535 nm and emission, 617 nm.

MTS Viability Assay

Cell proliferation was measured by using a CellTiter 96 AQnonradioactive cell proliferation assay (Promega). Cells were plated in96-well plates and grown until confluence. Peptides were added to afinal concentration of 5-100 μM. After incubation for 24 h at 37° C. (5%CO₂) MTS[3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]-phenozinemethosulfate solution (20 μl/well) was added and cells were againincubated for 2 h at 37° C. (5% CO₂). The MTS compound is bioreduced bycells into a colored formazan product that is soluble in tissue culturemedium. The quantity of the formazan product as measured by the amountof 490 nm absorbance is directly proportional to the number of livingcells in culture. Data are calculated as a percentage of the control(untreated) samples and represent the average of three wells in oneexperiment which was repeated three times per cell line.

Spectrofluorimetric Analysis of Caspase-3/7 Activity

5×10⁵ cells/ml were seeded into 96-well plate and grown overnight at 37°C. and 5% CO₂. Cells were incubated with different concentrations ofpeptide for 4 hours. Apo-ONE® caspase-3/7 reagent was added in a 1:1volume ratio and cells were incubated for 4 hours. Cells were thenanalyzed by fluorescence spectroscopy (GloMax®-Multi+ MicroplateMultimode Reader with Instinct™). Untreated cells were used as negativecontrol. Analysis was performed with Apo-ONE® Homogeneous Caspase-3/7Assay (see FIG. 7).

Hemolysis

The hemolytic activity towards human red blood cells (RBCs), which wereobtained from heparinized human blood, was determined by the release ofhemoglobin following one hour incubation at 37° C. in MHNA (MuellerHinton cation Non Adjusted). Percentage of hemolysis of RBCs wascalculated using 1% Triton as 100% lysis and PBS as 0% lysis, peptideconcentration was 500 μg/ml.

Results

In the present examples, toxicity, respectively selectivity of thepeptides of the present invention against melanoma, rhabdomyosarcoma andglioblastoma cell lines that expose the negatively charged lipidphosphatidylserine on the outside was examined. Selective peptides arenot toxic against normal non-tumor cells as melanocytes and fibroblastsor red blood cells in the same concentration range.

Selective and active peptides can be (retro-) isolated peptides(combination of the peptide moieties) of the present invention with orwithout linkers exhibiting defined secondary structures (as definedabove) and show cancer selective activity in vitro and in cancer modelsystems.

Exemplarily 4 peptides were chosen to represent the observed effects.The results present data gained on peptide PEP-322 and PEP-318,representative for peptide moieties but non-active peptides,R-DIM-P-PEP-322 representative for cancer active and specific peptidesand DIM-PEP-318 representative for cancer and non-cancer active,non-selective peptides (see Table 2).

Besides R-DIM-P-PEP-322 the peptides, DIM-PEP-322, R-DIM-PEP-322,R-DIM-PEP-316, R-DIM-PEP-323, DIM-PEP-324, R-DIM-P-PEP-324,R-DIM-P-PEP-332, R-DIM-P-PEP-334 were shown to be selective for cancercells.

TABLE 2 Overview of peptide sequences, net charge and hydrophobicityof the peptides examined Net ΔG_(wif) ^(a) sequence charge [kcal/mol]PEP-322 PFWRIRIRR  +5 n.d. R-DIM-P-PEP-322 PFWRIRIRRPRRIRIRWFP  +9-2.00/-2.60 PEP-318 FWQRRIRRWRR  +7 n.d. DIM-PEP-318FWQRRIRRWRRFWQRRIRRWRR +13 -8.17/-7.91 ^(a)Peptide hydrophobicityexpressed as transfer free energy of peptides from water to bilayerinterface (ΔG_(wif)) calculated from the whole-residue hydrophobicityscale taking into account the contribution of the C-terminal amide(Wimley et al., Biochemistry 35 (1996), 5109-5124) and the % helixgained from the respective CD data Since for the short peptides PEP-322and -318 the CD measurements are not accurate enough the ΔG_(wif) wasonly calculated for the isolated peptides.Peptide Structure—Activity and Selectivity

In Silico—Secondary Structure Prediction

“Isolated peptides” of several PEP peptides were first analyzed bysimulation of the secondary structure. The secondary structure ofputative membrane active isolated peptides were predicted by the onlineprogram PEP-FOLD:http://bioserv.rpbs.univ-paris-diderot.fr/PEP-FOLD/(Maupetit, J et al.Nucleic Acids Res. 37 (2009), W498-W503). From this analysis severalpeptides were selected for synthesis and activity studies according totheir high proportion of amphipathic β-sheet or α-helical structure.

The non-active peptide moieties partially turned out to be too short forassembly of a defined secondary structure. Interestingly the cancerspecific peptides (DIM-, R-DIM-, R-DIM-P-PEP-322, DIM-PEP-324 andR-DIM-PEP-316) formed 2β-strands or 2α-helices (R-DIM-PEP-316) with aturn in the middle and distribution of cationic and hydrophobic regions.For the active but non-selective peptide DIM-PEP-318 a linearamphipathic α-helix without a loop was predicted (see Table 3, FIG. 13).For human Lactoferricin (hLFcin) (37-61) containing a disulfide bridgevia Cys3 and Cys20 a structure with a helical and an extended partlinked via a loop was predicted which is in agreement with the structurereported for hLFcin in a membrane mimetic solvent (Gifford, J L et al.Cell. Mol. Life Sci. 62 (2005), 2588-2598).

TABLE 3 List of secondary structure prediction and cancer specificityfor isolated peptides (-retro); list includes all sequences in isolatedpeptide (DIM-) (peptide moiety sequence + peptide moiety sequence asdefined herein), isolated peptide retro (R-DIM-) (peptide moietysequence + peptide moiety retro sequence), isolated peptide with linkerX (DIM-X-) (peptide moiety sequence + X + peptide moiety sequence),isolated peptide retro with linker X (R-DIM-X-) (peptide moietysequence + X + peptide moiety retro sequence). H = helix, T = turn, β =β- strand. For positive or negative peptide specificity for tumor overnon tumor cells in the case of studied peptides the respective -foldspecificity for melanoma (SBcl2 or A375) over non tumor skin cells(melanocytes or normal human dermal fibroblasts (NHDF-c)) at 20 μMpeptide concentration after 8 hours incubation is listed (derived by PIuptake studies). Peptide moieties Secondary Structure Prediction Cancer(parents) DIM- DIM-X- R-DIM- R-DIM-X- Specificity PEP H 2H T H 2H TPEP-313 H T 2β T H H T β PEP-314 2H 2β T 3β 2T 2β T PEP-315 H 2H T H 2HT PEP-316 H T 2H T 2H T 2H T Yes (15fold)(R- DIM-) PEP-317 H 2H T H 2H TPEP-318 H 2H T H 2H T No (<1fold)(DIM-) PEP-319 2H T 2H T H 2H T PEP-320H 2H T 2H T 2H T PEP-322 2β T 2β T 2β T 2β T yes (50fold) (DIM-), 20fold(R-DIM-), >100 fold) (R-DIM- X-) PEP-215 2β T 2β T 2β T 2β T PEP-227 2H2H T H 2H T PEP-323 2 β T 2β T 2β T 4β T PEP-324 2β T 2β T 2β T 2β T yes(7fold) (DIM-), (15fold)(R- DIM-X-) PEP-325 2H T 2H T H 2H T PEP-326 2HT 2H T H 2H T PEP-327 2H T 2H T H 2H T PEP-328 2H 2H T H 2H T PEP-329 2H2H T H 2H T PEP-330 2H 2H T H 2H T PEP-331 2H 2H T H 2H T PEP-332 2H 2HT H 2H T (5fold)(R-DIM- X-) PEP-333 2H 2H T H 2 H T PEP-334 2β T 2β T 2βT 2β T (15fold)(R- DIM-X-) PEP-335 2H 2H T H 2H T PEP-336 H 2H T H 2H TPEP-337 2H T 2H T H 2H T weak (2fold)(R- DIM-) PEP-338 2β T 2β T =DIM-=DIM-X- PEP-339 2β T 2β T =DIM- =DIM-X-Model Studies

Circular Dichroism Spectroscopy—Secondary Structure Vs. Activity andSelectivity

Strikingly the selective peptide R-DIM-P-PEP-322 (FIG. 1B) exhibits asignificant increase of β-sheet conformation in the presence of thecancer mimic SDS, α-helical content is even further decreased. Moreoverthe structure of the peptide in the presence of the healthy mimic DPC isthe same as in solution, giving further hint for the cancer selectivetoxicity of these peptides (see also FIG. 2).

Percentage secondary structure calculations were done using Dichroweb,CDSSR Convolution Program using reference set 4 (Whitmore et al.,Nucleic Acids Res. 32 (2004), W668-W67; Whitmore et al., Biopolymers 89(2008), 392-400). The α-helical content is shown in dark gray at thebottom; β-turns in light grey; turns in middle grey; random coilstructures in dark grey at the top.

FIG. 2 now presents the results of circular dichroism spectroscopy ofthe non-specific peptide DIM-PEP-318 in contrast to the specific peptideR-DIM-PEP-322. In contrast to the PEP-322 peptide group, DIM-PEP-318possesses a higher α-helical content in the presence of the cancer mimicSDS as well as in the presence of the non-cancer mimic DPC. DIM-PEP-318adopts up to 75% α-helical structure without discrimination betweencancer and non-cancer cell mimic.

In Vitro Studies—Membrane Permeabilization

PI-Uptake—Increase of Activity by Sequence Doubling

Cytotoxic activity of the peptides towards melanoma cells of primary(SBcl-2) and metastatic lesions (WM164) and differentiatednon-tumorigenic melanocytes was determined by measurement of PI-uptake,which only occurs when integrity of the cell membrane is lost. Cellswere incubated in media containing serum for 8 h in the presence ofpeptides. Peptide concentrations were varied from 10 to 80 μM. FIG. 3illustrates that the peptide moiety PEP-322 is only minor active againstthe melanoma cell line SBcl-2 with less than 5% killing at a peptideconcentration of 80 μM, as well as against melanocytes with a moderatetwo-fold selectivity for WM164 cells at 20 μM peptide concentration(FIG. 3C). The isolated peptide by combination of the peptide moietywith its retro sequence and a Pro linker R-DIM-P-PEP-322 shows stronglyincreased activity against SBcl-2 compared to the its peptide moietywith very high specificity for the melanoma cell line. Already at apeptide concentrations of 20 μM, R-DIM-P-PEP-322 yields more than 80% PIpositive SBcl-2 cells, while only less than 1% of differentiatednon-tumorigenic melanocytes are killed (FIG. 3B), exhibiting aspecificity more than 100-fold for cancer cells (see FIG. 3C). Thesecond melanoma cell line, WM164, tested at 20 μM R-DIM-P-PEP-322peptide concentration is also highly sensitive for the peptide.

PI-Uptake—Specificity and Time Dependence of Killing-Correlation ofSpecificity; with Structure

Cytotoxic activity of the peptides towards melanoma cells of primary(SBcl-2) and metastatic lesions (WM164), a rhabdomyosarcoma cell line(TE671) and their healthy counterparts differentiated non-tumorigenicmelanocytes and normal human dermal fibroblasts (NHDF) was determined bymeasurement of PI-uptake, which indicates a loss of cell membraneintegrity (FIGS. 4 and 5). Cells were incubated in media containingserum for up to 8 h in the presence of peptides at 20 μM peptideconcentration.

Both peptide moieties PEP-322 as well as PEP-318 are only minor activeagainst cancer cells (<30%, see FIG. 4A-C). However, PEP-318 is evenslightly active against non-cancer cells, killing up to ˜30% of NHDF.Interestingly, the combination of the peptide moiety PEP-322 and itsretro sequence in form of the isolated peptide R-DIM-P-PEP-322 showshigh cancer toxicity (up to 80%) but with negligible non-cancer toxicity(see FIG. 4D-E). In contrast to DIM-PEP-318, R-DIM-P-PEP-322 kills quiteslowly reaching its highest activity not before 4-8 h. The secondpeptide moiety combination, namely DIM-PEP-318, possesses the highestand fastest anticancer activity with up to 90% killing within minutes(FIG. 4A-C). However, DIM-PEP-318 reveals to be quite unspecific sinceit is also highly active against differentiated non-tumorigenicmelanocytes as well as against normal human dermal fibroblasts (FIG.4D-E). Cytotoxic activity of the peptides after 8 h of incubation isgiven in FIG. 11. As can be seen in FIG. 12 all peptides with structurepredictions of an α-helix without a loop exhibit toxicity againstnon-tumor and tumor cell lines. The parent peptide hLFcin (37-61)comprising contrary to the peptides claimed here within a disulfide bondvia two cysteins (Cys3 and Cys20) shows only weak activity against thetested melanoma cell line A375 and glioblastoma cell line U87 mg and notoxicity against normal human dermal fibroblasts NHDF-c.

As shown in FIGS. 14 and 15 the isolated peptides show high cytotoxicityagainst the cell lines of malignant melanoma (A375) and glioblastoma(U87 mg) after 8 hours of incubation at 20 μM peptide concentration. Thecytotoxicity is very much improved compared to that exhibited by theparent peptide h LFcin. These two cancer types exhibit so far very poortreatability and bad prognosis.

Cell Viability—MTS Cell Proliferation

To determine long-time toxicity of peptides, a MTS cell proliferationassay was used to elucidate cell viability upon 24 h incubation withvariant peptide concentration and human melanoma cell lines SBcl-2 andWM164 and non-differentiated human skin fibroblast cell line NHDF. Asshown in FIG. 6A PEP-322 affects cancer cells and non-cancer cells onlymarginally even at higher peptide concentrations of 100 μM sustainingmore than 70% cell viability (IC_(50 WM164 and NHDF)>100 μM) and 40%(IC_(50 SBcl-2) 90). Combination of peptide moieties could highlyimprove anticancer activity. R-DIM-P-PEP-322 exhibits a decreased IC₅₀value of 8 μM and 15 μM for melanoma cell line SBcl-2 and melanomametastasis WM164, respectively (FIG. 6B) compared to an IC₅₀ value of 80μM for the non-cancer cell line, yielding 8-5-fold selectivity forcancer cells. DIM-PEP-318 shows also high activity against cancer cellsresulting in an IC₅₀<10 μM (FIG. 6C). As indicated already by PI uptakestudies this peptide is as toxic for non-cancer cells, revealed by avery low IC₅₀ of 10 μM for NHDF, as well. Results of MTS and PI assaywith cancer cells correlated well. An overview of IC₅₀ values is givenin Table 6.

TABLE 6 Comparison of IC₅₀ values determined through PI-uptake (8 h) andMTS cell viability assay (24 h). SBcl-2 (PI/MTS) Fibroblasts (PI/MTS)PEP-322 >80 μM/90 μM  >>80 μM/>100 μM R-DIM-P-PEP-322 8 μM/8 μM >>80μM/80 μM  PEP-318 n.d./n.d. n.d./n.d. DIM-PEP-318 <20 μM/6 μM  <20 μM/10μM 

Hemolytic Activity Against Red Blood Cells—Specificity

Hemolytic activity of peptides against red blood cells was tested at 500μg/ml peptide and 2.5% red blood cell concentration. It was verysurprising that DIM-PEP-318 was not hemolytic, considering the hightoxicity towards melanocytes and fibroblasts.

TABLE 7 Hemolytic activity of peptides against human red blood cells %lysis of 2.5% RBCs^(a) IC₅₀ [μg/ml] PEP-322 2^(b)  >500^(b)R-DIM-P-PEP-322 0.84 + 0.63 >500 PEP-318 n.d. n.d. DIM-PEP-318 2.87 +0.57 >500 ^(a)Percentage of hemolysis of human red blood cells (RBCs)was calculated following one hour incubation at 37° C. in PBS using 1%Triton X-100 as 100% lysis and PBS as 0% lysis, peptide concentrationwas 500 μg/ml. ^(b)from Zweytick et al., J. Biol. Chem. 286 (2011),.n.d. not determinedCaspase-3 Cleavage-Apoptosis or Necrosis

To clearly differentiate between necrotic and apoptotic killing acaspase-3/7 activity assay was used to detect emergence of apoptosis(FIG. 7). R-DIM-P-PEP-322 showed apoptosis indicated by a strongincrease of caspase-3/7 activity from the time-point of 4 hours ofincubation of melanoma cells SBcl-2 with 10-20 μM peptide, indicated bya 200-fold increase of green fluorescence. The non-specific peptideDIM-PEP-318 showed much lower caspase-3/7 activity, indicating anon-apoptotic killing mechanism like necrosis shown by a strongPI-uptake by the peptide (FIG. 8-11).

Additionally, apoptotic like blebbing of the cell membrane is observedduring incubation of the rhabdomyosarcoma cell line TE671 in thepresence of the peptide.

TABLE 8 Correlation of activity exhibited by peptides PEP-322 andR-DIM-P-PEP-322 in model and in vitro studies. R-DIM-P-  Peptide PEP-322PEP-322 amino acid sequence PFWRIRIRR-(NH2) -PRRIRIRWFP-NH₂ net charge+5 +9 cancer mimic /healthy mimic bilayer perturbation- +/- +++/-Differential Scanning Calorimetry permeability-ANTS/DPX +/- +++/-leakage bilayer affinity-quenching ++/- ++/- structure-CD >β-sheet/ as in > β-sheet/as in solution solutioncancer cells/healthy cells toxicity-PI uptake, MTT -/- +++/-cancer specificity (+) ++++

TABLE 9Correlation of activity exhibited by selective peptide R-DIM-P-PEP-322and non-selective peptide DIM-PEP-318 in model and in vitro studies.Peptide R-DIM-P-PEP-322 DIM-PEP-318 amino acid sequence PFWRIRIRR-P-FWQRRIRRWRR-  RRIRIRWFP-NH₂ FWQRRIRRWRR-NH₂ net charge +9 +13cancer mimic/healthy mimic bilayer perturbation- +++/- +++/+++Differential Scanning Calorimetry permeability-leakage +++/- +++/+bilayer affinity- ++/- ++/+ quenching structure-CD > β-sheets/as in >α-helical/> α-helical solution cancer cells/healthy cellstoxicity-PI uptake, MTT +++/- +++/+++ cancer specificity ++++ -Discussion

In this example the selective antitumor activity of the peptides of thepresent invention could be demonstrated. The peptide moieties(containing no disulfide bridge) derived of the membrane active part ofhLFcin such as PEP-322 and hLFcin (37-61) (one disulfide bridge) itselfexhibited only weak activity against melanoma cancer cell lines, thecombination of peptide moieties in R-DIM-P-PEP-322 (comprising 2beta-strands separated by a turn) showed highly increased activity.PI-uptake of melanoma cells upon incubation with peptide R-DIM-P-PEP-322further demonstrates that the peptide operates via a membrane mediatedway, since PI can only be taken up by cells that suffer membranedisintegration. Improved interaction of the isolated peptide with thecancer mimic PS correlated with increased activity against the melanomacancer cell line and non-interaction with the healthy mimic PCcorrelated with non-toxicity against non-cancer melanocytes. Theisolated peptide exhibits a high membrane destabilization emphasized byhighly increased membrane permeability of PS bilayers. Besides,permeability studies show that a certain threshold concentration of theisolated peptide is needed for induction of sufficient leakage ofANTS/DPX, differentiating it from highly lytic but mostly unspecificpeptides like melittin. In agreement also the effect on neutral lipidsis negligible. Moreover by calorimetric studies it could be demonstratedthat the effect of the isolated peptide is even much higher than that ofthe peptide moiety at doubled concentration, rather suggesting astructural effect than a simple mass and charge effect.

Trp localization studies of peptides showed that if a peptide is activeagainst a certain membrane, it exhibits a significant blue shift of Trpemission wavelength upon interaction with the membrane indicating a morehydrophobic environment of Trp due to interaction with the membraneinterface. In the case of the peptide moiety PEP-322 and combination ofpeptide moieties R-DIM-P-PEP-322 the blue shift is only detected inpresence of the target lipid PS present on the surface of cancermembranes, whereas in the presence of PC no blue shift appears, goinghand-in-hand with a selective toxicity against cancer cells in vitro.These findings are in line with the ability of Trp quenching, which isstrongly decreased only in the presence of the target lipid PS.Non-selective peptides like DIM-PEP-318 however reveal a blue shift inthe presence of both model systems.

Further structural information on the studied peptides was given by CDexperiments. Again structural changes for PEP-322 and the isolatedpeptide appear only in the presence of the negatively charged cancermimic (SDS). The peptide DIM-PEP-318 changes its structure inenvironment of both models conform to its low specificity. Only thenon-selective peptide shows an increase of the α-helical content in thepresence of both model systems, differently PEP-322 and R-DIM-P-PEP-322show an increase of the β-sheet content upon presence of the cancermodel SDS.

From the differences in activity displayed by the peptide moiety and thecombination thereof in the isolated peptide it was however surprisingthat both peptides show quite similar structural characteristics insolution and model system. Considering the shortness of the moietyPEP-322, it is even questionable if a β-sheet conformation is possible.It is moreover reasonable that two moiety peptide stretches arrange onthe lipid surface like a dimer, but not covalently linked. The combinedmoieties in the isolated peptide however are fixed in this conformationvia peptide bond and will create stronger membrane perturbance andfinally higher membrane permeabilization, which can explain its highlyincreased activity in model and cell system.

On the one hand it was demonstrated that high membrane interaction ofthe isolated peptides derived from the membrane active peptide PEP withanionic PS correlates with high activity against melanoma cells, on theother hand it could be shown that increasing interaction with thehealthy mimic neutral PC correlates with decreased specificity indicatedby increased interaction with non-cancer cell types like melanocytes orfibroblasts. This was demonstrated for the combination of two peptidemoieties PEP-318, namely DIM-PEP-318 also originally derived from PEP.The short peptide moieties PEP-322 and PEP-318 are only minor activeagainst cancer cells even if incubation time is extended to 8 h orhigher peptide concentrations are used. Partially the low activity canalso be due to less defined structure. In contrast, the combination ofthese peptide moieties in DIM-PEP-322 and DIM-PEP-318 exhibit increasedanticancer activity. However the different peptides seem to operate bydifferent mechanisms, since peptide DIM-PEP-318 reaches its maximumtoxicity against cancer cells already after 15 minutes, whereascontrariwise R-DIM-PEP-322 kills much slower reaching its maximumactivity not before 8 hours. Nevertheless it shows similar or evenincreased cancer toxicity compared to DIM-PEP-318, after long timeperiod. The different time dependence of cell killing by the peptidesindicates 2 different killing mechanism. The very fast action ofDIM-PEP-318 gives strong evidence for a direct membranolytic effectcausing necrosis. Prediction of the secondary structure of the peptidesproposes an amphiphatic α-helix. Structural analysis through CDspectroscopy of DIM-PEP-318 also reveals induction of a mostly α-helicalstructure in the presence of the cancer as well as the non-cancer mimicresulting in non-selective lysis of cells. The selective peptideR-DIM-P-PEP-322 obviously acts via a different mechanism. The relativelyslow action together with the observation of membrane blebbing andCaspase-3/7 activity is an indication for membrane-mediated apoptosis.For induction of apoptosis the peptide has to enter the cellspecifically over probably the PS compartments on the surface andfurther reach another negatively charged target on the surface of cancercell mitochondria, like cardiolipin. Successive swelling of mitochondriaand release of cytochrome-C activate the caspase dependent pathway ofthe programmed cell death. Interestingly R-DIM-P-PEP-322 shows inductionof an increase in the predominant β-sheet structure with a turn and nochanges in structure in the presence of the non-cancer cell mimic. Inthe secondary structure prediction an arrangement of 2β-strands withhydrophobic endings with a loop in the middle composed of cationic aminoacids was predicted. According to other prediction studies theamphipathic distribution of amino acids with a loop between 2β-strandsor 2α-helices seem to be important structural features for an active andspecific peptide.

The invention claimed is:
 1. A method of treating cancer comprisingadministering an isolated peptide that is 12 to 50 amino acids in lengthcomprising at least two peptide moieties, wherein said at least twopeptide moieties are: (a) at least two beta-strands; (b) at least twoalpha-helices; or (c) at least one beta-strand and at least onealpha-helix, wherein said beta-strands and/or alpha-helices areseparated from each other by at least one turn; wherein the peptide hasa net positive charge of +7 or more; wherein the peptide moieties havean amino acid sequence, selected from the group consisting of FWQRIRKVR(SEQ ID No. 1), FWQRRIRKVRR (SEQ ID No. 2), FWQRKIRKVRK (SEQ ID No. 3),FWQRNIRIRR (SEQ ID No. 4), FWQRNIRKVR (SEQ ID No. 5), FWQRNIRVR (SEQ IDNo. 6), FWQRNIRKVRR (SEQ ID No. 7), FWQRNIRKVKK (SEQ ID No. 8),FWQRNIRKVRRR (SEQ ID No. 9), FWQRNIRKVKKK (SEQ ID No. 10), FWQRNIRKVRRRR(SEQ ID No. 11), FWQRNIRKVRRRI (SEQ ID No. 12), FWQRNIRKVKKKK (SEQ IDNo. 13), FWQRNIRKVKKKI (SEQ ID No. 14), FWQRNIRKIR (SEQ ID No. 15),FWQRNIRKLR (SEQ ID No. 16), FWQRNIRKWR (SEQ ID No. 17), FWQRNWRKVR (SEQID No. 18), FWQRNFRKVR (SEQ ID No. 19), FWQRNYRKVR (SEQ ID No. 20),FWQRNIRKVS (SEQ ID No. 21), FWQRRIRIRR (SEQ ID No. 22), FWQRPIRKVR (SEQID No. 23), FWQRRIRKWR (SEQ ID No. 24), FWPRNIRKVR (SEQ ID No. 26),FWARNIRKVR (SEQ ID No. 27), FWIRNIRKVR (SEQ ID No. 28), FWLRNIRKVR (SEQID No. 29), FWVRNIRKVR (SEQ ID No. 30), FWQRNIFKVR (SEQ ID No. 31),FWQRNIYKVR (SEQ ID No. 32), FAWQRNIRKVR (SEQ ID No. 33), FIWQRNIRKVR(SEQ ID No. 34), FLWQRNIRKVR (SEQ ID No. 35), FVWQRNIRKVR (SEQ ID No.36), FWRIRKWR (SEQ ID No. 37), FWRIRKVR (SEQ ID No. 38), FWRWRR (SEQ IDNo. 39), FWRRWRR (SEQ ID No. 40), FWRRWIRR (SEQ ID No. 41), FWRGWRIRR(SEQ ID No. 42), FWRRFWRR (SEQ ID No. 43), FWRWRWR (SEQ ID No. 44),FWRIWRWR (SEQ ID No. 45), FWRIWRIWR (SEQ ID No. 46), FWRNIRKWR (SEQ IDNo. 47), FWRRRIRIRR (SEQ ID No. 48), FIWRWRWR (SEQ ID No. 49), PFWRWRIWR(SEQ ID No. 50), PFWRIRIRR (SEQ ID No. 51), PFWRQRIRR (SEQ ID No. 52),PFWRARIRR (SEQ ID No. 53), PFWRKRIRR (SEQ ID No. 54), PFWRKRLRR (SEQ IDNo. 55), PFWRKRWRR (SEQ ID No. 56), PFWRRRIRR (SEQ ID No. 57), PFWRRRWRR(SEQ ID No. 58), PFWRIRIRRD (SEQ ID No. 59), PFFWRIRIRR (SEQ ID No. 60),PWRIRIRR (SEQ ID No. 61), RFWQRNIRKVRR (SEQ ID No. 62), RFWQRNIRKYR (SEQID No. 63), PFWQRNIRKWR (SEQ ID No. 64), RFRWQRNIRKYRR (SEQ ID No. 65),RWKRINRQWF (SEQ ID No. 66), KRFCFKK (SEQ ID No. 67), KRFSFKKC (SEQ IDNo. 68), KRWSWKK (SEQ ID No. 69), FRFSFKK (SEQ ID No. 70), RRFWFRR (SEQID No. 71), FWRNIRIRR (SEQ ID No. 72), FWQRIRIRR (SEQ ID No. 73),FWRWRIWR (SEQ ID No. 74), FWRIRIRR (SEQ ID No. 75), FWRNIRIWRR (SEQ IDNo. 76) and FWRNIRIRR (SEQ ID No. 77), RFWQRNIRIRR (SEQ ID No. 78),RWQRNIRIRR (SEQ ID No. 79), RRIRINRQWF (SEQ ID No. 80), PFWRRQIRR (SEQID No. 81), PFWRKKLKR (SEQ ID No. 82), PWRRIRR (SEQ ID No. 83), PWRRKIRR(SEQ ID No. 84), PFWRRIRIRR (SEQ ID No. 85), and RRWFWRR (SEQ ID No.86), FWQRRIRRWRR (SEQ ID No. 112), PFWRRRIRIRR (SEQ ID No. 193),RRWFFWRR (SEQ ID No. 198) and the reverse sequences thereof; and whereinsaid peptide is devoid of intramolecular disulfide bonds; and whereinthe cancer is selected from the group consisting of melanoma,rhabdomyosarcoma, glioblastoma, colorectal cancer, breast cancer,lymphoma, prostate cancer, pancreatic cancer, renal cancer, ovariancancer, and lung cancer.
 2. The method of claim 1, wherein the peptideis selected from the group consisting of PFWRIRIRRXRRIRIRWFP (SEQ ID.No. 128), PWRIRIRRXRRIRIRWP (SEQ ID No. 184), RWKRINRQWFFWQRNIRKWR (SEQID. No. 106), PFWRIRIRRPFWRIRIRR (SEQ ID. No. 125), PFFWRIRIRRPFFWRIRIRR(SEQ ID. No. 141) and PFWRIRIRRRRIRIRWFP (SEQ ID. No. 127).
 3. Themethod of claim 2, wherein the peptide is selected from the groupconsisting of SEQ ID. Nos. 128, 184 and 106, wherein “X” is proline(Pro) and/or glycine (Gly)₁₋₃.
 4. The method of claim 1, wherein thepeptide is devoid of cysteine residues.
 5. The method of claim 1,wherein the isolated peptide comprises at least two peptide moietieshaving the same amino acid sequence, wherein the at least two peptidemoieties are fused directly or via a linker to each other.
 6. The methodof claim 5, wherein the isolated peptide comprises two peptide moietieshaving the same amino acid sequence, wherein the at least two peptidemoieties are fused directly or via a linker to each other.
 7. The methodof claim 1, wherein the isolated peptide comprises at least two peptidemoieties, wherein the at least one second peptide moiety is the reversesequence thereof, wherein the at least two peptide moieties are fuseddirectly or via a linker to each other.
 8. The method of claim 7,wherein the isolated peptide comprises two peptide moieties, wherein theat least one second peptide moiety is the reverse sequence thereof,wherein the at least two peptide moieties are fused directly or via alinker to each other.
 9. The method of claim 1, wherein the cancer ismelanoma, rhabdomyosarcoma, or glioblastoma cancer.
 10. The method ofclaim 1, wherein the peptide moieties have an amino acid sequence,selected from the group consisting of FWQRNIRIRR (SEQ ID No. 4),FWQRNIRKWR (SEQ ID No. 17), FWQRRIRKWR (SEQ ID No. 24), FWRRFWRR (SEQ IDNo. 43), FWRNIRKWR (SEQ ID No. 47), PFWRIRIRR (SEQ ID No. 51), PFWRQRIRR(SEQ ID No. 52), PFWRARIRR (SEQ ID No. 53), PFWRKRIRR (SEQ ID No. 54),PFWRKRLRR (SEQ ID No. 55), PFWRKRWRR (SEQ ID No. 56), PFWRRRIRR (SEQ IDNo. 57), PFWRRRWRR (SEQ ID No. 58), PFWRIRIRRD (SEQ ID No. 59),PFFWRIRIRR (SEQ ID No. 60), PWRIRIRR (SEQ ID No. 61), PFWQRNIRKWR (SEQID No. 64), RWKRINRQWF (SEQ ID No. 66), FWRIRIRR (SEQ ID No. 75),RRIRINRQWF (SEQ ID No. 80), PFWRRQIRR (SEQ ID No. 81), PFWRKKLKR (SEQ IDNo. 82), PWRRIRR (SEQ ID No. 83), PWRRKIRR (SEQ ID No. 84), RRWFWRR (SEQID No. 86), PFWRRRIRIRR (SEQ ID No. 193), and RRWFFWRR (SEQ ID No. 198),and the reverse sequences thereof.
 11. The method of claim 10, whereinthe cancer is melanoma, rhabdomyosarcoma, or glioblastoma cancer.